Glucagon superfamily peptides exhbiting G protein coupled receptor activity

ABSTRACT

Provided herein are glucagon superfamily peptides conjugated with GPCR ligands that are capable of acting at a G protein-coupled receptor. Also provided herein are pharmaceutical compositions and kits of the conjugates of the invention. Further provided herein are methods of treating a disease, e.g., a metabolic disorder, such as diabetes and obesity, comprising administering the conjugates of the invention.

Incorporated by reference in its entirety is a computer-readable aminoacid sequence listing submitted concurrently herewith and identified asfollows: One 875,196 byte ASCII (Text) file named “44632B_SeqListing,”created on Feb. 12, 2014.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application serial No. PCT/US2011/035912 filed May 10,2011, which claims priority to U.S. Provisional Patent Application No.61/334,433 filed May 13, 2010. The entire disclosures ofPCT/US2011/035912 and U.S. Ser. No. 61/334,433 are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION Field of the Disclosure

This invention provides glucagon superfamily peptides conjugated to Gprotein-coupled receptor ligands that are capable of acting at Gprotein-coupled receptors.

BRIEF DESCRIPTION OF RELATED TECHNOLOGY

Peptide-based drugs are highly effective medicines with a relativelyshort duration of action and variable therapeutic index. Activation ofseveral G protein-coupled receptors (GPCRs), for example, GPR40, GPR119,GRP120, and GPR30 has been demonstrated to produce favorable effects onglucose homeostasis, food intake, body weight gain and possibly alsoβ-cell preservation. Free fatty acids (FFAs) appear to stimulate insulinsecretion through direct islet GPCR signaling and indirect stimulationof incretin secretion via receptor-mediated mechanisms. GPR40 and GPR119are among the several receptors to play an important physiological rolein insulin release. It is now known that both GPR40 and GPR119 areexpressed in pancreatic β-cells and that activating these receptorsstimulates insulin secretion in a glucose-dependent manner GPR120 is areceptor for unsaturated fatty acids, such as α-linoleic acid. Itsexpression has been found in both intestinal L cells and islets.Stimulation of GPR120 has been demonstrated to increase insulin levelsand proliferation of β-cells. GPR30, a functional estrogen receptor, hasbeen shown to be involved in glucose homeostasis and reduction of bodyweight in the DIO model in rodents. Compounds capable of activatingthese above-mentioned receptors could prove valuable agents for thetreatment of type 2 diabetes and obesity. However, the ligandspreviously identified are generally low affinity agonists and may be oflimited therapeutic use.

Pre-proglucagon is a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofpre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide thatcorresponds to amino acids 72 through 108 of pre-proglucagon.GLP-1(7-36) amide or GLP-1(7-37) acid are biologically potent forms ofGLP-1, that demonstrate essentially equivalent activity at the GLP-1receptor.

Glucagon is used in the acute treatment of severe hypoglycemia.Oxyntomodulin has been reported to have pharmacological ability tosuppress appetite and lower body weight. GLP-1 and GLP-1 receptoragonists are used as treatment for Type II diabetes. Exendin-4 is apeptide present in the saliva of the Gila monster that resembles GLP-1in structure, and like glucagon and GLP-1, increases insulin release.

Gastric inhibitory polypeptide (GIP) is also known as aglucose-dependent insulinotropic peptide, and is a member of thesecretin family of hormones. GIP is derived from a 153-amino acidproprotein encoded by the GIP gene and circulates as a biologicallyactive 42-amino acid peptide. The GIP gene is expressed in the smallintestine as well as the salivary glands and is a weak inhibitor ofgastric acid secretion. In addition to its inhibitory effects in thestomach, in the presence of glucose, GIP enhances insulin release bypancreatic beta islet cells when administered in physiological doses.GIP is believed to function as an enteric factor that stimulates therelease of pancreatic insulin and that may play a physiological role inmaintaining glucose homeostasis.

Osteocalcin is a noncollagenous protein found in bone and dentin. It issecreted by osteoblasts and thought to play a role in mineralization andcalcium ion homeostasis. Osteocalcin has also been reported to functionas a hormone in the body, causing beta cells in the pancreas to releasemore insulin, and at the same time directing fat cells to release thehormone adiponectin, which increases sensitivity to insulin.

SUMMARY OF THE INVENTION

Provided herein are glucagon superfamily peptides conjugated to Gprotein-coupled receptor ligands (“GPCR ligands”), preferably GPCRligands involved in metabolism, including lipid metabolism or glucosehomeostasis. These conjugates with plural activities are useful for thetreatment of a variety of diseases.

The glucagon superfamily peptide conjugates of the invention can berepresented by the following formula:Q-L-Ywherein Q is a glucagon superfamily peptide, Y is a GPCR ligand, and Lis a linking group or a bond.

The glucagon superfamily peptide (Q) in some embodiments can be aglucagon related peptide that exhibits agonist activity at the glucagonreceptor, agonist activity at the GLP-1 receptor, agonist activity atthe GIP receptor, co-agonist activity at the glucagon and GLP-1receptors, co-agonist activity at the glucagon and GIP receptors,co-agonist activity at the GLP-1 and GIP receptors, or tri-agonistactivity at the glucagon, GIP, and GLP-1 receptors. In some embodiments,the glucagon related peptide exhibits antagonist activity at theglucagon, GLP-1 or GIP receptor. The activity of the glucagon relatedpeptide at the glucagon receptor, at the GLP-1 receptor, or at the GIPreceptor can be in accordance with any of the teachings set forthherein. In some specific embodiments, the glucagon related peptideexhibits at least 0.1% activity of native glucagon at the glucagonreceptor, at least 0.1% activity of native GLP-1 at the GLP-1 receptor,or at least 0.1% activity of native GIP at the GIP receptor.

The GPCR ligand (Y) is wholly or partly non-peptidic and acts at a Gprotein-coupled receptor with an activity in accordance with any of theteachings set forth herein. In some embodiments the GPCR ligand has anEC₅₀ or IC₅₀ of about 1 mM or less, or 100 μM or less, or 10 μM or less,or 1 μM or less. In some embodiments, the GPCR ligand has a molecularweight of up to about 5000 daltons, or up to about 2000 daltons, or upto about 1000 daltons, or up to about 500 daltons. The GPCR ligand mayact at any of the G protein-coupled receptors described herein or haveany of the structures described herein. In preferred embodiments, theGPCR is involved in metabolism, including lipid metabolism and glucosehomeostasis. In selected embodiments, the GPCR is a member of therhodopsin-like family, e.g. GPR30, GPR40, GPR119 or GPR120.

In some embodiments, the glucagon related peptide has an EC₅₀ (or IC₅₀)at the glucagon receptor within about 100-fold, or within about 75-fold,or within about 50-fold, or within about 40-, 30-, 25-, 20-, 15-, 10- or5-fold of the EC₅₀ or IC₅₀ of the GPCR ligand at its G protein-coupledreceptor. In some embodiments, the glucagon related peptide has an EC₅₀(or IC₅₀) at the GLP-1 receptor within about 100-fold, or within about75-fold, or within about 50-fold, or within about 40-, 30-, 25-, 20-,15-, 10- or 5-fold of the EC₅₀ or IC₅₀ of the GPCR ligand at its Gprotein-coupled receptor. In some embodiments, the glucagon relatedpeptide has an EC₅₀ (or IC₅₀) at the GIP receptor within about 100-fold,or within about 75-fold, or within about 50-fold, or within about 40-,30-, 25-, 20-, 15-, 10- or 5-fold of the EC₅₀ or IC₅₀ of the GPCR ligandat its G protein-coupled receptor.

In some aspects of the invention, prodrugs of Q-L-Y are provided whereinthe prodrug comprises a dipeptide prodrug element (A-B) covalentlylinked to an active site of Q via an amide linkage. Subsequent removalof the dipeptide under physiological conditions and in the absence ofenzymatic activity restores full activity to the Q-L-Y conjugate.

In some aspects of the invention, pharmaceutical compositions comprisingthe Q-L-Y conjugate and a pharmaceutically acceptable carrier are alsoprovided.

In other aspects of the invention, methods are provided foradministering a therapeutically effective amount of a Q-L-Y conjugatedescribed herein for treating a disease or medical condition in apatient. In some embodiments, the disease or medical condition isselected from the group consisting of metabolic syndrome, diabetes,obesity, liver steatosis, and a neurodegenerative disease.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 presents an alignment of the amino acid sequences of variousglucagon superfamily peptides or relevant fragments thereof. The aminoacid sequence presented are GHRH (SEQ ID NO: 1619), PHI (SEQ ID NO:1622), VIP (SEQ ID NO: 1620), PACAP-27 (SEQ ID NO: 1621), Exendin-4 (SEQID NO: 1618), GLP-1 (SEQ ID NO: 1603), Glucagon (SEQ ID NO: 1601),Oxyntomodulin (SEQ ID NO: 1679), GIP (SEQ ID NO: 1607), GLP-2 (SEQ IDNO: 1680) and Secretin (SEQ ID NO: 1624). The alignment shows how aminoacid positions of glucagon can correspond to amino acid positions inother glucagon superfamily peptides.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. Analternative desired effect for the glucagon peptides of the presentdisclosure would include treating hyperglycemia, e.g., as measured by achange in blood glucose level closer to normal, or inducing weightloss/preventing weight gain, e.g., as measured by reduction in bodyweight, or preventing or reducing an increase in body weight, ornormalizing body fat distribution. The amount that is “effective” willvary from subject to subject, depending on the age and general conditionof the individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route, e.g., subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein the term “patient” without further designation isintended to encompass any warm blooded vertebrate domesticated animal(including for example, but not limited to livestock, horses, cats, dogsand other pets), mammals, and humans.

The term “isolated” as used herein means having been removed from itsnatural environment. In some embodiments, the analog is made throughrecombinant methods and the analog is isolated from the host cell.

The term “purified,” as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment and means having been increased in purityas a result of being separated from other components of the originalcomposition. The term “purified polypeptide” is used herein to describea polypeptide which has been separated from other compounds including,but not limited to nucleic acid molecules, lipids and carbohydrates.

As used herein, the term “peptide” encompasses a sequence of 2 or moreamino acids and typically less than 50 amino acids, wherein the aminoacids are naturally occurring or coded or non-naturally occurring ornon-coded amino acids. Non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.“Non-coded” as used herein refer to an amino acid that is not anL-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,Trp, Tyr.

As used herein, “partly non-peptidic” refers to a molecule wherein aportion or all of the molecule is a chemical compound or substituentthat has biological activity and that does not comprises a sequence ofamino acids.

As used herein, “non-peptidic” refers to a molecule has biologicalactivity and that does not comprise a sequence of amino acids.

As used herein, the terms “polypeptide” and “protein” are terms that areused interchangeably to refer to a polymer of amino acids, withoutregard to the length of the polymer. Typically, polypeptides andproteins have a polymer length that is greater than that of “peptides.”In some instances, a protein comprises more than one polypeptide chaincovalently or noncovalently attached to each other.

Throughout the application, all references to a particular amino acidposition by number (e.g., position 28) refer to the amino acid at thatposition in native glucagon (SEQ ID NO: 1601) or the corresponding aminoacid position in any analogs thereof. For example, a reference herein to“position 28” would mean the corresponding position 27 for an analog ofglucagon in which the first amino acid of SEQ ID NO: 1601 has beendeleted. Similarly, a reference herein to “position 28” would mean thecorresponding position 29 for a analog of glucagon in which one aminoacid has been added before the N-terminus of SEQ ID NO: 1601.

As used herein an “amino acid modification” refers to (i) a substitutionor replacement of an amino acid of the reference peptide (e.g. SEQ IDNOs: 1601, 1603, 1607) with a different amino acid (naturally-occurringor coded or non-coded or non-naturally-occurring amino acid), (ii) anaddition of an amino acid (naturally-occurring or coded or non-coded ornon-naturally-occurring amino acid) to the reference peptide (e.g. SEQID NOs: 1601, 1603, 1607) or (iii) a deletion of one or more amino acidsfrom the reference peptide (e.g. SEQ ID NOs: 1601, 1603, 1607).

In some embodiments, the amino acid substitution or replacement is aconservative amino acid substitution, e.g., a conservative substitutionof the amino acid at one or more of positions 1, 2, 5, 7, 8, 10, 11, 12,13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29. As used herein, theterm “conservative amino acid substitution” is the replacement of oneamino acid with another amino acid having similar properties, e.g.,size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, andincludes exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negative-charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positive-charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

In some embodiments, the amino acid substitution is not a conservativeamino acid substitution, e.g., is a non-conservative amino acidsubstitution.

As used herein the term “amino acid” encompasses any molecule containingboth amino and carboxyl functional groups, wherein the amino andcarboxyl groups are attached to the same carbon (the alpha carbon). Thealpha carbon optionally may have one or two further organicsubstituents. For the purposes of the present disclosure designation ofan amino acid without specifying its stereochemistry is intended toencompass either the L or D form of the amino acid, or a racemicmixture. However, in the instance where an amino acid is designated byits three letter code and includes a superscript number (i.e., Lys⁻¹),such a designation is intended to specify the native L form of the aminoacid, whereas the D form will be specified by inclusion of a lower cased before the three letter code and superscript number (i.e., dLys⁻¹).

As used herein the term “hydroxyl acid” refers to an amino acid that hasbeen modified to replace the alpha carbon amino group with a hydroxylgroup.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that has a negative charge (i.e.,deprotonated) or positive charge (i.e., protonated) in aqueous solutionat physiological pH. For example negative-charged amino acids includeaspartic acid, glutamic acid, cysteic acid, homocysteic acid, andhomoglutamic acid, whereas positive-charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 coded amino acids, as well as atypical ornon-naturally occurring or non-coded amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety (other than the alpha carboxylic acidof the amino acid), including for example, a side chain carboxylic acidor sulfonic acid group.

As used herein an “acylated” amino acid is an amino acid comprising anacyl group which is non-native to a naturally-occurring amino acid,regardless by the means by which it is produced. Exemplary methods ofproducing acylated amino acids and acylated peptides are known in theart and include acylating an amino acid before inclusion in the peptideor peptide synthesis followed by chemical acylation of the peptide. Insome embodiments, the acyl group causes the peptide to have one or moreof (i) a prolonged half-life in circulation, (ii) a delayed onset ofaction, (iii) an extended duration of action, (iv) an improvedresistance to proteases, such as DPP-IV, and (v) increased potency atthe glucagon superfamily peptide receptor.

As used herein, an “alkylated” amino acid is an amino acid comprising analkyl group which is non-native to a naturally-occurring amino acid,regardless of the means by which it is produced. Exemplary methods ofproducing alkylated amino acids and alkylated peptides are known in theart and including alkylating an amino acid before inclusion in thepeptide or peptide synthesis followed by chemical alkylation of thepeptide. Without being held to any particular theory, it is believedthat alkylation of peptides will achieve similar, if not the same,effects as acylation of the peptides, e.g., a prolonged half-life incirculation, a delayed onset of action, an extended duration of action,an improved resistance to proteases, such as DPP-IV, and increasedpotency at the glucagon superfamily peptide receptor.

The term “C₁-C_(n) alkyl” wherein n can be from 1 through 18, as usedherein, represents a branched or linear alkyl group having from one tothe specified number of carbon atoms. For example, C₁-C₆ alkylrepresents a branched or linear alkyl group having from 1 to 6 carbonatoms. Typical C₁-C₁₈ alkyl groups include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl and the like. Alkyl groups optionally can besubstituted, for example, with hydroxy (OH), halo, aryl, carboxyl, thio,C₃-C₈ cycloalkyl, and amino.

The term “C₀-C_(n) alkyl” wherein n can be from 1-18, as used herein,represents a branched or linear alkyl group having up to 18 carbonatoms. For example, the term “(C₀-C₆ alkyl)OH” represents a hydroxylparent moiety attached to an alkyl substituent having up to 6 carbonatoms (e.g. —OH, —CH₂OH, —C₂H₄OH, —C₃H₆OH, —C₄H₈OH, —C₅H₁₀OH, —C₆H₁₂OH).

The term “C₃-C_(n) cycloalkyl” wherein n can be from 1 through 10, asused herein, represents a cyclic alkyl group having from one to thespecified number of carbon atoms. For example, C₃-C₈ cycloalkylrepresents a cyclic alkyl group having from 3 to 8 carbon atoms. TypicalC₃-C₁₀ cyclo alkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and the like. Cycloalkyl groupsoptionally can be substituted, for example, with hydroxy (OH), halo,aryl, carboxyl, thio, and amino.

The term “C₂-C_(n) enyl” wherein n can be from 2 through 18, as usedherein, represents an unsaturated branched or linear group having from 2to the specified number of carbon atoms and at least one double bond.Examples of such groups include, but are not limited to, 1-propenyl,2-propenyl (—CH₂—CH═CH₂), 1,3-butadienyl, (—CH═CHCH═CH₂), 1-butenyl(—CH═CHCH₂CH₃), hexenyl, pentenyl, and the like. Alkenyl groupsoptionally can be substituted, for example, with hydroxy (OH), halo,aryl, carboxyl, thio, C₃-C₈ cycloalkyl, and amino.

The term “C₂-C_(n) ynyl” wherein n can be from 2 to 18, refers to anunsaturated branched or linear group having from 2 to n carbon atoms andat least one triple bond. Examples of such groups include, but are notlimited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,and the like. Alkynyl groups optionally can be substituted, for example,with hydroxy (OH), halo, aryl, carboxyl, thio, C₃-C₈ cycloalkyl, andamino.

As used herein the term “aryl” refers to a monocyclic or polycyclic (forexample, bicyclic, tricyclic, or tetracyclic) aromatic groups. The sizeof the aryl ring or rings is indicated by designating the number ofcarbons present. For example, the term “(C₁-C₃ alkyl)(C₆-C₁₀ aryl)”refers to a 6 to 10 membered aryl that is attached to a parent moietyvia a one to three membered alkyl chain. Unless otherwise indicated, anaryl group can be unsubstituted or substituted with one or more, and inparticular one to five groups independently selected from, for example,halo, alkyl, alkenyl, OCF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, C₃-C₈cycloalkyl, C(O)Oalkyl, aryl, and heteroaryl. Exemplary aryl groupsinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,chlorophenyl, indanyl, indenyl, methylphenyl, methoxyphenyl,trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, and thelike.

As used herein, the term “heteroaryl” refers to a monocyclic orpolycyclic ring system containing one or more aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring. The size of the heteroaryl ring and the presence of substituentsor linking groups are indicated by designating the number of carbonspresent. For example, the term “(C₁-C₆ alkyl)(C₅-C₆ heteroaryl)” refersto a 5 or 6 membered heteroaryl that is attached to a parent moiety viaa one to 6 membered alkyl chain. Unless otherwise indicated, aheteroaryl group can be unsubstituted or substituted with one or more,and in particular one to five groups independently selected from, forexample, halo, alkyl, alkenyl, OCF₃, NO₂, CN, NC, OH, alkoxy, amino,CO₂H, C₃-C₈ cycloalkyl, C(O)Oalkyl, aryl, and heteroaryl. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl,triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

As used herein, the term “heteroalkyl” refers to a linear or branchedhydrocarbon containing the indicated number of carbon atoms and at leastone heteroatom in the backbone of the structure. Suitable heteroatomsfor purposes herein include but are not limited to N, S, and O.Heteroalkyl groups optionally can be substituted, for example, withhydroxy (OH), halo, aryl, carboxyl, and amino

As used herein, the term “heterocycloalkyl” refers to a cyclichydrocarbon containing the indicated number of carbon atoms and at leastone heteroatom in the backbone of the structure. Suitable heteroatomsfor purposes herein include but are not limited to N, S, and O.Heterocycloalkyl groups optionally can be substituted, for example, withhydroxy (OH), halo, aryl, carboxyl, and amino

As used herein, the term “halogen” or “halo” refers to one or moremembers of the group consisting of fluorine, chlorine, bromine, andiodine.

As used herein, the term “glucagon related peptide” refers to thosepeptides which have biological activity (as agonists or antagonists) atany one or more of the glucagon, GLP-1, GLP-2, and GIP receptors andcomprise an amino acid sequence that shares at least 40% sequenceidentity (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%)with at least one of native glucagon, native oxyntomodulin, nativeexendin-4, native GLP-1, native GLP-2, or native GIP. Unless otherwisestated, any reference to an amino acid position in a glucagon relatedpeptide (e.g. for linkage of a GPCR ligand, a conjugate moiety, ahydrophilic polymer, acylation or alkylation) refers to thecorresponding position relative to the native glucagon amino acidsequence (SEQ ID NO: 1601).

As used herein, the term “selectivity” of a molecule for a firstreceptor relative to a second receptor refers to the following ratio:EC₅₀ of the molecule at the second receptor divided by the EC₅₀ of themolecule at the first receptor. For example, a molecule that has an EC₅₀of 1 nM at a first receptor and an EC₅₀ of 100 nM at a second receptorhas 100-fold selectivity for the first receptor relative to the secondreceptor.

The term “identity” as used herein relates to the similarity between twoor more sequences. Identity is measured by dividing the number ofidentical residues by the total number of residues and multiplying theproduct by 100 to achieve a percentage. Thus, two copies of exactly thesame sequence have 100% identity, whereas two sequences that have aminoacid deletions, additions, or substitutions relative to one another havea lower degree of identity. Those skilled in the art will recognize thatseveral computer programs, such as those that employ algorithms such asBLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol.Biol. 215:403-410) are available for determining sequence identity

As used herein, the term “glucagon superfamily peptide” refers to agroup of peptides related in structure in their N-terminal andC-terminal regions (see, for example, Sherwood et al., Endocrine Reviews21: 619-670 (2000)). Members of this group include all glucagon relatedpeptides, as well as Growth Hormone Releasing Hormone (GHRH; SEQ ID NO:1619), vasoactive intestinal peptide (VIP; SEQ ID NO: 1620), pituitaryadenylate cyclase-activating polypeptide 27 (PACAP-27; SEQ ID NO: 1621),peptide histidine isoleucine (PHI; SEQ ID NO: 1642), peptide histidinemethionine (PHM; SEQ ID NO: 1622), Secretin (SEQ ID NO: 1623), andanalogs, derivatives or conjugates with up to 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 amino acid modifications relative to the native peptide. Suchpeptides preferably retain the ability to interact (agonist orantagonist) with receptors of the glucagon receptor superfamily. Unlessotherwise stated, any reference to an amino acid position in a glucagonsuperfamily peptide (e.g. for linkage of a GPCR ligand, a conjugatemoiety, a hydrophilic polymer, acylation or alkylation) refers to thecorresponding position relative to the native glucagon amino acidsequence (SEQ ID NO: 1601), see FIG. 1 for an alignment ofrepresentative glucagon superfamily peptides.

The term “glucagon agonist peptide” refers to a compound that binds toand activates downstream signaling of the glucagon receptor. However,this term should not be construed as limiting the compound to havingactivity at only the glucagon receptor. Rather, the glucagon agonistpeptides of the present disclosures may exhibit additional activities atother receptors, as further discussed herein. Glucagon agonist peptides,for example, may exhibit activity (e.g., agonist activity) at the GLP-1receptor and/or the GIP receptor. Also, the term “glucagon agonistpeptide” should not be construed as limiting the compound to onlypeptides. Rather, compounds other than peptides are encompassed by thisterm. Accordingly, the glucagon agonist peptide in some aspects is apeptide in conjugate form (a heterodimer, a multimer, a fusion peptide),a chemically-derivatized peptide, a pharmaceutical salt of a peptide, apeptidomimetic, and the like.

The term “GLP-1 agonist peptide” refers to a compound that binds to andactivates downstream signaling of the GLP-1 receptor. However, this termshould not be construed as limiting the compound to having activity atonly the GLP-1 receptor. Rather, the GLP-1 agonist peptides of thepresent disclosures may exhibit additional activities at otherreceptors, as further discussed herein. GLP-1 agonist peptides, forexample, may exhibit activity (e.g., agonist activity) at the glucagonreceptor and/or the GIP receptor. Also, the term “GLP-1 agonist peptide”should not be construed as limiting the compound to only peptides.Rather, compounds other than peptides are encompassed by this term.Accordingly, the GLP-1 agonist peptide in some aspects is a peptide inconjugate form (a heterodimer, a multimer, a fusion peptide), achemically-derivatized peptide, a pharmaceutical salt of a peptide, apeptidomimetic, and the like.

The term “GIP agonist peptide” refers to a compound that binds to andactivates downstream signaling of the GIP receptor. However, this termshould not be construed as limiting the compound to having activity atonly the GIP receptor. Rather, the GIP agonist peptides of the presentdisclosures may exhibit additional activities at other receptors, asfurther discussed herein. GIP agonist peptides, for example, may exhibitactivity (e.g., agonist activity) at the GLP-1 receptor. Also, the term“GIP agonist peptide” should not be construed as limiting the compoundto only peptides. Rather, compounds other than peptides are encompassedby this term. Accordingly, the GIP agonist peptide in some aspects is apeptide in conjugate form (a heterodimer, a multimer, a fusion peptide),a chemically-derivatized peptide, a pharmaceutical salt of a peptide, apeptidomimetic, and the like.

The term “glucagon antagonist peptide” refers to a compound thatcounteracts glucagon activity or prevents glucagon function. Forexample, a glucagon antagonist exhibits at least 60% inhibition (e.g.,at least 70%, 80%, 90% or more inhibition) of the maximum responseachieved by glucagon at the glucagon receptor. In a specific embodiment,the glucagon antagonist at a concentration of about 1 μM exhibits lessthan about 20% of the maximum agonist activity achieved by glucagon atthe glucagon receptor (e.g. less than about 10% or 5%). This term shouldnot be construed as limiting the compound to having activity at only theglucagon receptor. Rather, the glucagon antagonist peptides of thepresent disclosures may exhibit additional activities at the glucagonreceptor (e.g., partial agonism) or other receptor. Glucagon antagonistpeptides, for example, may exhibit activity (e.g., agonist activity) atthe GLP-1 receptor. Also, the term “glucagon antagonist peptide” shouldnot be construed as limiting the compound to only peptides. Rather,compounds other than peptides are encompassed by these terms.Accordingly, in some aspects, the glucagon agonist peptide is a peptidein conjugate form, a chemically-derivatized peptide, a pharmaceuticalsalt of a peptide, a peptidomimetic, and the like.

The term “GLP-1 antagonist peptide” refers to a compound thatcounteracts GLP-1 activity or prevents GLP-1 function. For example, aGLP-1 antagonist exhibits at least 60% inhibition (e.g., at least 70%,80%, 90% or more inhibition) of the maximum response achieved by GLP-1at the GLP-1 receptor. In a specific embodiment, a GLP-1 antagonist at aconcentration of about 1 μM exhibits less than about 20% of the maximumagonist activity achieved by GLP-1 at the GLP-1 receptor (e.g. less thanabout 10% or 5%). The term should not be construed as limiting thecompound to having activity at only the GLP-1 receptor. Rather, theGLP-1 antagonist peptides of the present disclosures may exhibitadditional activities at the GLP-1 receptor (e.g., partial agonism) orother receptor. GLP-1 antagonist peptides, for example, may exhibitactivity (e.g., agonist activity) at the glucagon receptor. Also, theterm “GLP-1 antagonist peptide” should not be construed as limiting thecompound to only peptides. Rather, compounds other than peptides areencompassed by these terms. Accordingly, in some aspects, the GLP-1agonist peptide is a peptide in conjugate form, a chemically-derivatizedpeptide, a pharmaceutical salt of a peptide, a peptidomimetic, and thelike.

The term “GIP antagonist peptide” refers to a compound that counteractsGIP activity or prevents GIP-1 function. For example, a GIP antagonistexhibits at least 60% inhibition (e.g., at least 70%, 80%, 90% or moreinhibition) of the maximum response achieved by GIP at the GIP receptor.In a specific embodiment, a GIP antagonist at a concentration of about 1μM exhibits less than about 20% of the maximum agonist activity achievedby GIP at the GIP receptor (e.g. less than about 10% or 5%). The termshould not be construed as limiting the compound to having activity atonly the GIP receptor. Rather, the GIP antagonist peptides of thepresent disclosures may exhibit additional activities at the GIPreceptor (e.g., partial agonism) or other receptor. GIP antagonistpeptides, for example, may exhibit activity (e.g., agonist activity) atthe glucagon receptor. Also, the term “GIP antagonist peptide” shouldnot be construed as limiting the compound to only peptides. Rather,compounds other than peptides are encompassed by these terms.Accordingly, in some aspects, the GIP agonist peptide is a peptide inconjugate form, a chemically-derivatized peptide, a pharmaceutical saltof a peptide, a peptidomimetic, and the like.

As used herein, the terms “glucagon analog” and “glucagon peptide” canbe used interchangeably to refer to an analog of glucagon that has theindicated activity at a glucagon related peptide receptor.

As used herein the term “native glucagon” refers to a peptide consistingof the sequence of SEQ ID NO: 1601.

As used herein, the term “native GLP-1” is a generic term thatdesignates GLP-1(7-36) amide (SEQ ID NO: 1603), GLP-1(7-37) acid (SEQ IDNO: 1604) or a mixture of those two compounds.

As used herein, the term “native GIP” refers to a peptide consisting ofSEQ ID NO: 1607.

As used herein, “glucagon potency” or “potency compared to nativeglucagon” of a molecule refers to the ratio of the EC₅₀ of the moleculeat the glucagon receptor divided by the EC₅₀ of native glucagon atglucagon receptor.

As used herein, “GLP-1 potency” or “potency compared to native GLP-1” ofa molecule refers to the ratio of the EC₅₀ of the molecule at GLP-1receptor divided by the EC₅₀ of native GLP-1 at GLP-1 receptor.

As used herein, “GIP potency” or “potency compared to native GIP” of amolecule refers to the ratio of the EC₅₀ of the molecule at the GIPreceptor divided by the EC₅₀ of native GIP at the GIP receptor.

As used herein, “GPCR ligand” refers to a hydrophobic or lipophilicmoiety that has biological activity (either agonist or antagonist) at aG protein-coupled receptor (GPCR). The GPCR ligand is wholly or partlynon-peptidic. In some embodiments, the GPCR ligand is an agonist thatbinds to and activates the GPCR. In other embodiments, the GPCR ligandis an antagonist. In some embodiments, the GPCR ligand is an antagonistthat acts by wholly or partially blocking binding of native ligand tothe active site. In other embodiments, the GPCR ligand is an antagonistthat acts by binding to the active site or an allosteric site andpreventing activation of, or de-activating, the GPCR.

As used herein, “steroids and derivatives thereof” refers to compounds,either naturally occurring or synthesized, having a structure of FormulaA:

wherein R¹ and R², when present, are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula A to a GPCR; R³ and R⁴ are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula A to a GPCR; and each dashed line represents an optionaldouble bond. Formula A may further comprise one or more substituents atone or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16,and 17 Contemplated optional substituents include, but are not limitedto, OH, NH₂, ketone, and C₁-C₁₈ alkyl groups. Specific, nonlimitingexamples of steroids and derivatives thereof include cholesterol, cholicacid estradiol, testosterone, and hydrocortisone.

As used herein, “fatty acids and derivatives thereof” refers tocarboxylic acids comprising a unbranched C₁ to C₂₈ alkyl or C₂ to C₂₈alkenyl moiety and can optionally comprise one or more halo substituentsand/or optionally comprise one or more substituents other than halo. Insome embodiments, the unbranched alkyl or alkenyl moiety can be whollyhalo substituted (e.g., all hydrogens replaced with halo atoms). A shortchain fatty acid comprises 1-5 carbon atoms. A medium chain fatty acidcomprises 6-12 carbon. A long chain fatty acid comprises 13-22 carbonatoms. A very long chain fatty acid comprises 23-28 carbon atoms.Specific, nonlimiting examples of fatty acids include formic acid,acetic acid, n-caproic acid, heptanoic acid, caprylic acid, nonanoicacid, capric acid, undecanoic acid, lauric acid, tridecanoic acid,myristic acid, pentadeconoic acid, palmitic acid, heptadecanoic acid,stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid,behenic acid, tricosanoic acid, mead acid, myristoleic acid, palmitoleicacid, sapienic acid, oleic acid, linoleic acid, α-linolenic acid,elaidic acid, petroselinic acid, arachidonic acid,dihydroxyeicosatetraenoic acid (DiHETE), octadecynoic acid,eicosatriynoic acid, eicosadienoic acid, eicosatrienoic acid,eicosapentaenoic acid, erucic acid, dihomolinolenic acid, docosatrienoicacid, docosapentaenoic acid, docosahexaenoic acid, and adrenic acid.

As used herein, “free fatty amides and derivatives thereof” refers tostructures of Formula C:

wherein n is 0-26 and each R⁵, when present, is independently a moietythat permits or promotes agonist or antagonist activity upon binding ofthe compound of Formula C to a GPCR. For example, each R⁵, when present,can independently be hydrogen, C₁-C₇ alkyl, or halogen; and R⁶ can behydrogen, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₀-C₈alkyl)halo, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, heteroalkyl, (C₀-C₈alkyl)aryl, or (C₀-C₈ alkyl)heteroaryl. Nonlimiting examples of freefatty amides include oleamide, stearoylethanolamide,palmitoylethanolamide, linolenoyl ethanolamide, linoleoyl ethanolamide,anandamide, oleoylehtanolamide, and N-oleoyldopamine.

As used herein, “phospholipids and derivatives thereof” refers tosaturated or unsaturated compounds of Formula D:

wherein n is 0-26, each R⁵, when present, R⁷, and R⁸ are moieties thatpermit or promote agonist or antagonist activity upon binding of thecompound of Formula D to a GPCR. For example, each R⁵, when present, canindependently be hydrogen, C₁-C₇ alkyl or halogen; R⁷ can be choline,ethanolamine, or inositol; and R⁸ can be hydrogen or

Nonlimiting examples of phospholipids and derivatives thereof includeplatelet activating factor, lyso-platelet activating factor, oleolysophosphatidylcholine, stearoyl lysophosphatidylcholine, palmitoyllysophosphatidylcholine, lysophosphatidylethanolamine, andlysophosphatidylinositol.

As used herein, “grifolic acid and derivatives thereof” refers tocompounds of Formula E:

wherein each R⁹ is independently a functional group that permits orpromotes agonist or antagonist activity upon binding of the compound ofFormula E to a GPCR. For example, each R⁹ can independently be hydrogen,OH, or O(C₁-C₈ alkyl).

As used herein, “diacetylphloroglucinol and derivatives thereof” refersto compounds of Formula F:

wherein each m is independently 0-15, and each R¹⁰ is independently afunctional group that permits or promotes agonist or antagonist activityupon binding of the compound of Formula F to a GPCR. For example, eachR¹⁰ can independently be hydrogen or C₁-C₁₈ alkyl.

As used herein, “retinoic acid and derivatives thereof” refers tocompounds of Formula G:

wherein R¹¹ is a functional group that permits or promotes agonist orantagonist activity upon the binding of the compound of Formula G to aGPCR, and

represents either E or Z stereochemistry. For example, R¹¹ can beC(O)OH, CH₂OH, or C(O)H. Nonlimiting examples of retinoic acid andderivatives thereof include all-trans-retinoic acid, retinol, retinal,and 11-cis-retinoic acid.

As used herein, “thiazolidinedione derivative” refers to moleculescomprising a functional group represented by Formula H:

Nonlimiting examples of thiazolidinedione derivatives includerosiglitazone, pioglitazone, and troglitazone.

As used herein, “linking group” is a molecule or group of molecules thatbinds two separate entities to one another. Linking groups may providefor optimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include hydrolyzable groups, photocleavable groups,acid-labile moieties, base-labile moieties and enzyme cleavable groups.

As used herein, the term “prodrug” is defined as any compound thatundergoes chemical modification before exhibiting its fullpharmacological effects.

As used herein, a “dipeptide” is the result of the linkage of an α-aminoacid or α-hydroxyl acid to another amino acid, through a peptide bond.

As used herein the term “chemical cleavage” absent any furtherdesignation encompasses a non-enzymatic reaction that results in thebreakage of a covalent chemical bond.

Embodiments

The present disclosures provide glucagon superfamily peptides conjugatedwith GPCR ligands. In some aspects, the GPCR ligands are capable ofacting at G protein-coupled receptors involved in metabolism or glucosehomeostasis, and the conjugate provides superior biological effects onmetabolism or glucose homeostasis compared to the peptide alone or theGPCR ligand alone. Without being bound by a theory of the invention, theGPCR ligand may serve to target the glucagon superfamily peptide toparticular types of cells or tissues; or alternatively the glucagonsuperfamily peptide may serve to target the GPCR ligand or enhance itstransport into the cell, e.g. through binding of peptide to a receptorthat internalizes the conjugate.

The glucagon superfamily peptide conjugates of the invention can berepresented by the following formula:Q-L-Ywherein Q is a glucagon superfamily peptide, Y is a GPCR ligand, and Lis a linking group or a bond.

The glucagon superfamily peptide (Q) in some embodiments can be aglucagon related peptide that exhibits agonist activity at the glucagonreceptor, agonist activity at the GLP-1 receptor, agonist activity atthe GIP receptor, co-agonist activity at the glucagon and GLP-1receptors, co-agonist activity at the glucagon and GIP receptors,co-agonist activity at the GLP-1 and GIP receptors, or tri-agonistactivity at the glucagon, GIP, and GLP-1 receptors. In some embodiments,the glucagon related peptide exhibits antagonist activity at theglucagon, GLP-1 or GIP receptor.

The glucagon superfamily peptide (Q) in some embodiments may be aglucagon-related peptide, Growth Hormone Releasing Hormone (GHRH; SEQ IDNO: 1619), vasoactive intestinal peptide (VIP; SEQ ID NO: 1620),Pituitary adenylate cyclase-activating polypeptide 27 (PACAP-27; SEQ IDNO: 1621), peptide histidine methionine (PHM; SEQ ID NO: 1622), orSecretin (SEQ ID NO: 1623), and/or and analogs, derivatives andconjugates thereof. Glucagon superfamily peptides may have commonstructural characteristics, including but not limited to homology withinthe N-terminal amino acids and/or alpha-helical structure within theC-terminal portion. It is believed that the C-terminus generallyfunctions in receptor binding and the N-terminus generally functions inreceptor signaling. A few amino acids in the N-terminal portion andC-terminal portion are highly conserved among members of the glucagonsuperfamily, for example, His1, Gly4, Phe6, Phe22, Val23, Trp25, andLeu26, with amino acids at these positions showing identity,conservative substitutions or similarity in amino acid side chains. Insome embodiments the glucagon related peptide Q is glucagon (SEQ ID NO:1601), oxyntomodulin (SEQ ID NO: 1606), exendin-4 (SEQ ID NO: 1618),Glucagon-like peptide-1 (GLP-1) (amino acids 7-37 provided as SEQ IDNOs: 1603 and 1604), Glucagon-like peptide-2 (GLP-2) (SEQ ID NO: 1608),GIP (SEQ ID NO: 1607) or analogs, derivatives and conjugates of theforegoing. In some embodiments Q as a glucagon related peptide comprisesan amino acid sequence that is at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the correspondingsequence of native glucagon, native oxyntomodulin, native exendin-4,native (7-37)GLP-1, native GLP-2, or native GIP over the length of thenative peptide (or over the positions which correspond to glucagon, seee.g., FIG. 1). In other embodiments, a glucagon superfamily peptide (Q)comprises an amino acid sequence of native glucagon, native exendin-4,native (7-37)GLP-1, native GLP-2, native GHRH, native VIP, nativePACAP-27, native PHM, native Oxyntomodulin, native Secretin, or nativeGIP with up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.In still further embodiments, Q comprises an amino acid sequence whichis a chimera of two or more native glucagon related peptide sequences.In some embodiments, Q comprises an amino acid sequence at least about50% identical to native glucagon (SEQ ID NO: 1601) that retains thealpha-helix conformation of the amino acids corresponding to amino acids12-29.

In related aspects, the invention provides peptide conjugatesrepresented by the formulaQ-L-Ywherein Q is osteocalcin, calcitonin, amylin, or an analog, derivativeor conjugate thereof, rather than a glucagon superfamily peptide; Y is aGPCR ligand; and L is a linking group or a bond. In some embodiments, Qcomprises osteocalcin (SEQ ID NO: 1644), or an amino acid sequence thatis at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,or 95% identical to native osteocalcin over the length of the nativepeptide. Q may comprise an analog of osteocalcin with up to 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid modifications relative to nativeosteocalcin, or a truncated analog of osteocalcin (e g, amino acids70-84) with up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidmodifications relative to the native truncated osteocalcin. In someembodiments, Q comprises calcitonin (SEQ ID NO: 1645), or an amino acidsequence that is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% identical to native calcitonin over the length ofthe native peptide. Q may comprise an analog of calcitonin with up to 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications relative to nativecalcitonin. In some embodiments, Q comprises amylin (SEQ ID NO: 1646),or an amino acid sequence that is at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to native amylinover the length of the native peptide. Q may comprise an analog ofamylin with up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidmodifications relative to native amylin.The GPCR Ligand (Y)

In the present disclosures relating to Q-L-Y conjugates, Y is a ligandthat acts at any G protein-coupled receptor (GPCR), including any onethe “G protein-coupled receptor superfamily” (GPCR superfamily), or aclass or subgroup thereof. The GPCR superfamily is composed of receptorstypically sharing a common structural motif of seven transmembranehelical domains. GPCRs play a crucial role in receiving chemical signalsfrom other cells and, in response, activating certain cellular responsessuch as, for example, cellular metabolism, cell growth and motility,inflammation, neuronal signaling, and blood coagulation.

GPCR ligands bind to the portion of the GPCR that is on the outside of acell. Ligand binding to the GPCR causes a conformational change in theGPCR, allowing the GPCR to act as a guanine nucleotide exchange factor.The ligand-bound GPCR activates an associated G protein by exchangingits bound GDP for a GTP. As a result, the G protein's alpha-subunit,together with the bound GTP, dissociate from the beta and gamma subunitsof the GPCR to initiate intracellular signaling proteins or to targetfunctional proteins. G protein-coupled receptors are important astargets for molecules such as hormones, neurotransmitters andphysiologically active substances, which control, regulate or adjust thefunctions of living bodies. For example, GPCRs include receptors forbiogenic amines, e.g., dopamine, epinephrine, histamine, glutamate(metabotropic effect), acetylcholine (muscarinic effect), and serotonin;for lipid mediators of inflammation such as prostaglandins, plateletactivating factor, and leukotrienes; for peptide hormones such ascalcitonin, C5a anaphylatoxin, follicle stimulating hormone,gonadotropin releasing hormone, neurokinin, oxytocin, and for proteasessuch as thrombin, trypsin, and factor VIIa/Xa; and for sensory signalmediators, e.g., retinal photopigments and olfactory stimulatorymolecules.

G protein-coupled receptors can be divided generally into six classesbased on sequence homology and functional similarity: Class A(Rhodopsin-like), Class B (Secretin receptor family), Class C(Metabotropic glutamate/pheromone), Class D (Fungal mating pheromonereceptors), Class E (Cyclic AMP receptors), and Class F(Frizzled/Smoothened).

Class A (Rhodopsin-like) GPCRs, for example, represent a protein familythat includes hormone, neurotransmitter and light receptors, all ofwhich transduce extracellular signals through interaction with guaninenucleotide-binding (G) proteins. Members of Class A receptors include:Melatonin receptor, Interleukin-8 receptor, Orexin receptor, AngiotensinII receptor, Chemokine receptor, Galanin receptor, Bradykinin receptor,Endothelin receptor, Histamine H2 receptor, Somatostatin receptor,Neuropeptide Y receptor, Urotensin II receptor, GPR orphan receptor,Olfactory receptor, Mas transmembrane protein, Formyl/methionyl peptidereceptor, Histamine H1 receptor, Dopamine receptor, Muscarinicacetylcholine receptor, C—X—C chemokine receptor type 5,5-Hydroxytryptamine 7 receptor, G10D orphan receptor,Prolactin-releasing peptide receptor, RDC1 orphan receptor, Opioidreceptor, 5-Hydroxytryptamine 4 receptor, Bombesin receptor, Adenosinereceptor, Gonadotrophin releasing hormone receptor, Melanocortin/ACTHreceptor, Neurokinin receptor, Opsin, Retinal pigment epithelium GPCR,Vasopressin receptor, Octopamine receptor, Glycoprotein hormonereceptor, P2Y5 purinoceptor, Cannabinoid receptor, 5-Hydroxytryptaminereceptor, 5-Hydroxytryptamine 6 receptor, Adrenergic receptor,Anaphylatoxin chemotactic receptor, DEZ orphan receptor, GPR1 orphanreceptor, GPR4 orphan receptor, Platelet-activating factor receptor, P2purinoceptor, Peropsin, APJ receptor, Growth hormone secretagoguereceptor type 1, GPR37 orphan receptor, Protease-activated receptor,Histamine H3 receptor, Leukotriene B4 receptor, Neurotensin receptor,Sphingosine 1-phosphate receptor, Lysophosphatidic acid receptor,Cysteinyl leukotriene receptor, CX3C Fractalkine chemokine receptor, G2Alysophosphatidylcholine receptor, OGR1 sphingosylphosphorylcholinereceptor, Neuromedin U receptor, XCR1 Lymphotactin chemokine receptor,Neuropeptide FF receptor, Psychosine receptor, Histamine H4 receptor,KISS-1 peptide receptor, Relaxin receptor, Melanin-concentrating hormonereceptor, Prostanoid receptor, Cholecystokinin receptor, Trace aminereceptor, Thyrotropin-releasing hormone receptor, Neuropeptide Wreceptor, GPR40-related receptor, and G-protein-coupled receptor 30.

GPR30 is a member of the rhodopsin-like family of G protein-coupledreceptors and has been demonstrated to be a functional estrogenreceptor. Female GPR30^((−/−)) mice exhibit hyperglycemia and impairedglucose tolerance, reduced body growth, increased blood pressure, andreduced serum IGF-I levels, demonstrating the importance of thisreceptor to normal metabolic function. GPR30 is capable of binding17β-estradiol (E2) in vitro with high affinity resulting in increases incAMP production and intracellular [Ca²⁺]. Thus, without wishing to bebound by any theory, it is believed that conjugates of the disclosureexhibiting GPR30 agonist activity are useful for treating hyperglycemicconditions, such as diabetes mellitus.

GPR40 is also a member of the rhodopsin-like family of GPCRs. It ishighly expressed in pancreatic β-cells and is activated by medium- andlong-chain saturated and unsaturated free fatty acids. In Chinesehamster ovary (CHO) cells, exogenously expressed GPR40 increasedintracellular [Ca²⁺] and activated extracellular signal-related kinase1/2. In rat pancreatic β-cells, linolenic acid reduced voltage-gated K⁺current through GPR40-mediated cAMP levels and protein kinase Aactivity, leading to enhanced β-cell excitability and insulin secretion.Thus, without wishing to be bound by any theory, it is believed thatconjugates of the disclosure exhibiting GPR40 agonist activity areuseful for treating hyperglycemic conditions, such as diabetes mellitus.

In humans GPR119, also a member of the rhodopsin-like family, is highlyexpressed in pancreatic islet β-cells and fetal liver. Phospholipids andfatty acid amides have been described as possible endogenous ligands forGPR119. Transfection of cells with GPR119 show an increase inconstitutive intracellular cAMP levels. When treated withplatelet-activating factor and lyso-platelet activating factor, CHOcells expressing GPR119 exhibit increased cAMP levels. Without wishingto be bound by any theory, it is believed that conjugates of thedisclosure exhibiting GPR119 agonist activity are useful for treatinghyperglycemic conditions, such as diabetes mellitus.

GPR120, also member of the rhodopsin-like family, is highly expressed inthe human intestinal tract and has been shown to be activated by medium-to long-chain free fatty acids, including α-linoleic acid. Stimulationof GPR120 has been demonstrated to increase insulin levels andproliferation of β-cells. Mouse enteroendocrine STC-1 cells secreteGLP-1 and cholecystokinin upon challenge with free fatty acids and thisresponse can be inhibited by transfection with an RNAi expression vectorspecific for GPR120, indicating a role in metabolic homestasis. Withoutwishing to be bound by any theory, it is believed that conjugates of thedisclosure exhibiting GPR120 agonist activity are useful for treatinghyperglycemic conditions, such as diabetes mellitus.

Activity of the GPCR Ligand

In some embodiments, Y exhibits an EC₅₀ for GPCR activation (or in thecase of an antagonist, an IC₅₀) of about 10 mM or less, or 1 mM (1000μM) or less (e.g., about 750 μM or less, about 500 μM or less, about 250μM or less, about 100 μM or less, about 75 μM or less, about 50 μM orless, about 25 μM or less, about 10 μM or less, about 7.5 μM or less,about 6 or less, about 5 μM or less, about 4 μM or less, about 3 μM orless, about 2 μM or less or about 1 μM or less). In some embodiments, Yexhibits an EC₅₀ or IC₅₀ at a GPCR of about 1000 nM or less (e.g., about750 nM or less, about 500 nM or less, about 250 nM or less, about 100 nMor less, about 75 nM or less, about 50 nM or less, about 25 nM or less,about 10 nM or less, about 7.5 nM or less, about 6 nM or less, about 5nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or lessor about 1 nM or less). In some embodiments, Y has an EC₅₀ or IC₅₀ at aGPCR which is in the picomolar range. Accordingly, in some embodiments,Y exhibits an EC₅₀ or IC₅₀ at a GPCR of about 1000 pM or less (e.g.,about 750 pM or less, about 500 pM or less, about 250 pM or less, about100 pM or less, about 75 pM or less, about 50 pM or less, about 25 pM orless, about 10 pM or less, about 7.5 pM or less, about 6 pM or less,about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pMor less).

In some embodiments, Y exhibits an EC₅₀ or IC₅₀ at a GPCR that is about0.1 pM or more, about 0.5 pM or more, or about 1 pM or more. Gprotein-coupled receptor activation (GPCR activity) can be measured invitro by any method known in the art, including measurement ofintracellular Ca²⁺ concentration as described by Hara et al.,Naunyn-Schmied Arch Pharmacol 380(3):247-55 (2009) or measurement ofintracellular cAMP concentration as described by Overton et al., CellMetabolism, 3, 165-175 (2006).

Briefly, to measure intracellular [Ca²⁺]_(i), cells expressing the GPCRof interest are seeded at a density of 2×10⁵ cells/well oncollagen-coated 96-well plates, incubated at 37° C. for 21 hours, andthen incubated in Hanks' balanced salt solution (HBSS, pH 7.4)containing Calcium Assay Kit Component A (Molecular Devices, Sunnyvale,Calif.) for one hour at room temperature. Mobilization of [Ca²⁺]_(i)evoked by agonists is monitored using a fluorometric imaging platereader.

To measure cAMP production, HEK293 cell monolayers expressing the GPCRof interest under the control of a tetracycline-inducible promoter arepretreated overnight with 10-20 ng/mL tetracycline to induce expressionof the GPCR. Treated monolayers are then incubated for 30 minutes at 37°C. with a particular agonist in stimulation buffer plus 1% DMSO. Cellsare then lysed and cyclic AMP content is determined by any method knownin the art, e.g., with the Perkin Elmer AlphaScreen cAMP kit.

In some embodiments, including any of the preceeding embodiments, Yexhibits about 0.001% or more, about 0.01% or more, about 0.1% or more,about 0.5% or more, about 1% or more, about 5% or more, about 10% ormore, about 20% or more, about 30% or more, about 40% or more, about 50%or more, about 60% or more, about 75% or more, about 100% or more, about125% or more, about 150% or more, about 175% or more, about 200% ormore, about 250% or more, about 300% or more, about 350% or more, about400% or more, or about 450% or higher activity at the GPCR relative tothe native ligand (GPCR potency). In some embodiments, Y exhibits about100% or less or about 500% or less activity at the GPCR relative tonative ligand. The activity of Y at a receptor relative to a nativeligand of the receptor is calculated as the inverse ratio of EC₅₀s for Yversus the native ligand. In some embodiments, Y is the native ligand ofthe receptor.

In some embodiments, Y exhibits an EC₅₀ or IC₅₀ at a GPCR that is aboutthat is about 0.1 pM or more, about 0.5 pM or more, or about 1 pM ormore, and about 100% or less or about 500% or less activity at the GPCRrelative to native ligand. In some embodiments, Y exhibits an EC₅₀ orIC₅₀ at a GPCR that is about that is about 0.1 pM or more and about 500%or less activity at the GPCR relative to native ligand.

Structure of the GPCR Ligand (Y)

The GPCR ligand of the invention (Y) is partly or wholly non-peptidicand is hydrophobic or lipophilic. In some embodiments, the GPCR ligandhas a molecular weight that is about 5000 daltons or less, or about 4000daltons or less, or about 3000 daltons or less, or about 2000 daltons orless, or about 1750 daltons or less, or about 1500 daltons or less, orabout 1250 daltons or less, or about 1000 daltons or less, or about 750daltons or less, or about 500 daltons or less, or about 250 daltons orless. The structure of Y can be in accordance with any of the teachingsdisclosed herein.

Y can exhibit any structure that enables it to act at any Gprotein-coupled receptor (GPCR), including any one of the “Gprotein-coupled receptor superfamily” (GPCR superfamily), or a class orsubgroup thereof. In some embodiments, Y acts at a Class A GPCR. Inthese embodiments, Y can be, for example, an adrenergic receptoragonist, such as a β3 adrenergic receptor agonist, which acts toincrease thermogenesis and resembles the effect of glucagon, or anicotinic acetylcholine receptor agonist, such as an α7-nicotinicacetylcholine receptor agonist, which plays a role in decreasingobesity-induced inflammation and improved metabolic syndrome (see Wanget al., “Activation of the Cholinergic Antiinflammatory PathwayAmeliorates Obesity-Induced Inflammation and Insulin Resistance”Endocrinology 152(3):836-845 (2011).

β3 adrenergic receptor agonists include amibegron(ethyl([(7S)-7-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)-5,6,7,8-tetrahydronaphthalen-2-yl]oxy)acetate),solabegron(3′-[(2-{[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}ethyl)amino]biphenyl-3-carboxylicacid), CL316,243 [Fu et al., (2008) “The effects of beta(3)-adrenoceptoragonist CL-316,243 on adiponectin, adiponectin receptors and tumornecrosis factor-alpha expressions in adipose tissues of obese diabeticKKAy mice.” European journal of pharmacology 584 (1): 202-6], L-796,568[Larsen et al., (2002) “Effect of a 28-d treatment with L-796568, anovel β3-adrenergic receptor agonist, on energy expenditure and bodycomposition in obese men”. The American Journal of Clinical Nutrition 76(4): 780-8], and LY-368,842, Ro40-2148,(S)-6-(4-(2-((3-(9H-carbazol-4-yloxy)-2-hydroxypropyl)amino)-2-methylpropyl)phenoxy)-3-pyridinecarboxamidemonohydrochloride,(S)-4-{2-[2-hydroxy-3-(4-hydroxyphenoxyl)propylamino]ethyl}phenoxymethylcyclohexylphosphinicacid lithium salt,4-{1-[2-(S)-hydroxy-3-(4-hydroxyphenoxy)-propylamino]cyclopentylmethyl)phenyl}-phosphonicacid lithium salt,(RR)-5-{2-[2-(3,4-dihydroxyphenyl)-2-hydroxyethylamino]propyl}-1,3-benzodioxole-2,2-dicarboxylate[Arch, (2002) “β₃-adrenoceptor agonists: potential, pitfalls andprogress”. Eur J Pharm 440:99-107], and analogs of any of the foregoingthat retain the 133 adrenergic receptor agonist activity, includingpharmaceutically acceptable salts of such compounds. All references areand β3 adrenergic receptor agonists disclosed therein are incorporatedby reference in their entirety.

α7-nicotinic acetylcholine receptor agonists include(+)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzo[b]furan-2-carboxamide,A-582941, AR-R17779((25)-2′H-spiro[4-azabicyclo[2.2.2]octane-2,5′-[1,3]oxazolidin]-2′-one),TC-1698, TC-5619(N-[(2S,3S)-2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]-1-benzofuran-2-carboxamide),GTS-21(3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine),PHA-543,613(N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide),PNU-282,987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide),PHA-709829, SSR-180,711 ((4-bromophenyl)1,4-diazabicyclo[3.2.2]nonane-4-carboxylate), tropisetron, WAY-317,538(5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide),anabaseine, choline and nicotine, and analogs of any of the foregoingthat retain the a7-nicotinic acetylcholine receptor agonist activity,including pharmaceutically acceptable salts of such compounds.

In the embodiments described herein, Y is conjugated to L (e.g. when Lis a linking group) or Q (e.g. when L is a bond) at any position of Ythat is capable of reacting with Q or L. One skilled in the art couldreadily determine the position and means of conjugation in view ofgeneral knowledge and the disclosure provided herein.

In any of the embodiments described herein wherein Y comprises atetracyclic skeleton having three 6-membered rings joined to one5-membered ring or a variation thereof (e.g. a Y that acts at thevitamin D receptor), the carbon atoms of the skeleton are referred to byposition number, as shown below:

For example, a modification having a ketone at position-6 refers to thefollowing structure:

In some embodiments of the invention, Y acts at any GPCR. In theseembodiments, Y can have any structure that permits or promotes agonistactivity upon binding to a GPCR, while in other embodiments Y is anantagonist of a GPCR.

Steroid or Derivative Thereof

In some embodiments of the invention, Y is a steroid or derivativethereof. In exemplary embodiments, Y comprises a structure as shown inFormula A:

wherein R¹ and R², when present, are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula A to a GPCR; R³ and R⁴ are independently moieties that permitor promote agonist or antagonist activity upon binding of the compoundof Formula A to a GPCR; and each dashed line represents an optionaldouble bond. Formula A may further comprise one or more substituents atone or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16,and 17. Contemplated optional substituents include, but are not limitedto, OH, NH₂, ketone, and C₁-C₁₈ alkyl groups.

In some embodiments, Y comprises a structure of Formula A wherein

R¹ is present and is hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl,(C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈ alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl, (C₀-C₈ alkyl)C(O)O heteroaryl,(C₀-C₈ alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, or SO₃H;

R² is present and is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, C₂-C₁₈ alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈alkyl)heteroaryl, (C₀-C₈ alkyl)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC₂-C₁₈alkenyl, (C₀-C₈ alkyl)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH,(C₀-C₈alkyl)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈alkyl)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₁₈alkyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkynyl,(C₀-C₈ alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl,(C₀-C₈ alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl,(C₀-C₈ alkyl)C(O)O heteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)OC(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈alkyl)NR²⁴C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈alkyl)NR²⁴C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈alkyl)OC(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)OC(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)OH, (C₀-C₈alkyl)OC(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈alkyl)NR²⁴(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₁₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(O)OH;

R³ is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₁₈alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀-C₈ alkyl)NR²⁴C₁-C₁₈alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkynyl,(C₀-C₈alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl,(C₀-C₈ alkyl)C(O)O heteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)OC(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈alkyl)NR²⁴C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈alkyl)NR²⁴C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈alkyl)OC(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)OC(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)OH, (C₀-C₈alkyl)OC(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈alkyl)NR²⁴(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₁₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(O)OH;

R⁴ is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₁₈alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀-C₈ alkyl)NR²⁴C₁-C₁₈alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkynyl,(C₀-C₈alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl,(C₀-C₈ alkyl)C(O)O heteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)OC(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈alkyl)NR²⁴C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈alkyl)NR²⁴C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈alkyl)OC(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)OC(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)OH, (C₀-C₈alkyl)OC(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈alkyl)NR²⁴(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₁₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(O)OH; and

R²⁴ is hydrogen or C₁-C₁₈ alkyl.

In some embodiments, Y comprises a structure of Formula A wherein

R¹ is present and is hydrogen, C₁-C₇ alkyl; (C₀-C₃ alkyl)C(O)C₁-C₇alkyl, (C₀-C₃ alkyl)C(O)aryl, or SO₃H;

R² is present and is hydrogen, halo, OH, or C₁-C₇ alkyl;

R³ is hydrogen, halo, OH, or C₁-C₇ alkyl;

R⁴ is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂₋₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)OC₁-C₈ alkyl, (C₀-C₈ alkyl)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₈alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀-C₈ alkyl)NR²⁴C₁-C₈ alkyl,(C₀-C₈ alkyl)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkynyl, (C₀-C₈alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₈alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(O)H, (C₀-C₈alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈ alkyl)C(O)OC₁-C₈alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₈ alkynyl,(C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl, (C₀-C₈ alkyl)C(O)Oheteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₈ alkyl, (C₀-C₈ alkyl)OC(O)C₂-C₈alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴C₁-C₈alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₈alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈ alkyl)C(O)NR²⁴aryl, (C₀-C₈alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈ alkyl)NR²⁴C(O)C₁-C₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈ alkyl)NR²⁴C(O)C₂-C₈ alkynyl,(C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈ alkyl)OC(O)OC₁-C₈ alkyl, (C₀-C₈alkyl)OC(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(O)OC₂-C₈ alkynyl, (C₀-C₈alkyl)OC(O)OH, (C₀-C₈ alkyl)OC(O)NR²⁴C₁-C₈ alkyl, (C₀-C₈alkyl)OC(O)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₈ alkynyl,(C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈ alkyl)NR²⁴(O)OC₁-C₈ alkyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₈ alkynyl, or(C₀-C₈ alkyl)NR²⁴(O)OH; and,

R²⁴ is hydrogen or C₁-C₇ alkyl.

In some embodiments, R¹ is hydrogen, propionate, acetate, benzoate, orsulfate; R² is hydrogen or methyl; R³ is hydrogen or methyl; and R⁴ isacetate, cypionate, hemisucciniate, enanthate, or propionate.

In embodiments wherein Y comprises a structure of Formula A, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula A that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula A and means of conjugation of Formula A to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula A is conjugated to L or Q at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofFormula A. In some embodiments, Formula A is conjugated to L or Q atposition 1, 3, 6, 7, 12, 10, 13, 16, 17, or 19 of Formula A.

Free Fatty Acid or Derivative Thereof

In some embodiments of the invention, Y is a free fatty acid orderivative thereof, as shown in Formula B:

wherein n is 0-26 and each R⁵, when present, are each independently amoiety that permits or promotes agonist or antagonist activity uponbinding of the compound of Formula B to a GPCR. In some embodiments, Ycomprises a structure of Formula B, wherein n is 0-26 and each R⁵ isindependently hydrogen, C₁-C₇ alkyl, or halogen. In some embodimentsFormula B is saturated such as, for example, formic acid, acetic acid,n-caproic acid, heptanoic acid, caprylic acid, nonanoic acid, capricacid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid,pentadeconoic acid, palmitic acid, heptadecanoic acid, stearic acid,nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid,tricosanoic acid, and derivatives thereof. In some embodiments Formula Bis unsaturated with either cis or trans stereochemistry such as, forexample, mead acid, myristoleic acid, palmitoleic acid, sapienic acid,oleic acid, linoleic acid, α-linolenic acid, elaidic acid, petroselinicacid, arachidonic acid, dihydroxyeicosatetraenoic acid (DiHETE),octadecynoic acid, eicosatriynoic acid, eicosadienoic acid,eicosatrienoic acid, eicosapentaenoic acid, erucic acid, dihomolinolenicacid, docosatrienoic acid, docosapentaenoic acid, docosahexaenoic acid,adrenic acid, and derivatives thereof.

In some embodiments Formula B is a saturated or unsaturated short chainfatty acid, wherein n is 0-3. In some embodiments, Formula B is asaturated or unsaturated medium chain fatty acid, wherein n is 4-10. Insome embodiments, Formula B is a saturated or unsaturated long chainfatty acid, wherein n is 11-20. In some embodiments, Formula B is asaturated or unsaturated very long chain fatty acid, wherein n is 21-26.

In some embodiments, the tail of Formula B is further modified tocomprise a moiety such as a carboxylic acid, a carboxylic acidderivative, or amide. For example, a modified compound of Formula B caninclude MEDICA16, as shown below:

In embodiments wherein Y comprises a structure of Formula B, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula B that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula B and means of conjugation of Formula B to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula B is conjugated to L or Q at any position onFormula B. In some embodiments, Formula B is conjugated to L or Qthrough a terminal carboxylic acid moiety.

Free Fatty Amide or Derivative Thereof

In some embodiments of the invention, Y is a free fatty amide orderivative thereof, as shown in Formula C:

wherein n is 0-26 and each R⁵, when present, is independently a moietythat permits or promotes agonist or antagonist activity upon binding ofthe compound of Formula C to a GPCR. In some embodiments, Y comprises astructure of Formula C, wherein n is 0-26; each R⁵, when present, isindependently hydrogen, or C₁-C₇ alkyl, halogen; and R⁶ is hydrogen,C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₀-C₈ alkyl)halo,C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, or(C₀-C₈ alkyl)heteroaryl. In some embodiments Formula C is saturated,while in other embodiments, Formula C is unsaturated with either cis ortrans stereochemistry.

In some embodiments, Y comprises a structure of Formula C, wherein n is4-26; each R⁵, when present, is hydrogen; and R⁶ is hydrogen. Forexample, Y can include oleamide, as shown below:

In some embodiments, Y comprises a structure of Formula C, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; andR⁶ is (C₁-C₈ alkyl)OH. Nonlimiting examples of compounds of Formula Cwhen R⁶ is (C₁-C₈ alkyl)OH include:

and derivatives thereof.

In some embodiments, Y comprises a structure of Formula C, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; andR⁶ is (C₁-C₃ alkyl)aryl. For example, Y can include N-oleoyldopamine, asshown below:

In embodiments wherein Y comprises a structure of Formula C, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula C that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula C and means of conjugation of Formula C to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula C is conjugated to L or Q at any position onFormula C. In some embodiments, Formula C is conjugated to L or Qthrough a terminal carboxylic acid or through a hydroxyl moiety.

Phospholipid or Derivative Thereof

In some embodiments of the invention, Y is a phospholipid or derivativethereof, as shown in Formula D:

wherein n is 0-26, each R⁵, R⁷, and R⁸ are moieties that permit orpromote agonist or antagonist activity upon binding of the compound ofFormula D to a GPCR. In some embodiments, Y comprises a structure ofFormula D, wherein n is 0-26; each R⁵, when present, is independentlyhydrogen, C₁-C₇ alkyl, or halogen; R⁷ is choline, ethanolamine, orinositol; and R⁸ is hydrogen or

In some embodiments Formula D is saturated, while in other embodiments,Formula D is unsaturated with either cis or trans stereochemistry.

In some embodiments, Y comprises a structure of Formula D, wherein eachn is independently 4-26; each R⁵, when present, is independentlyhydrogen or halogen; R⁷ is choline; and R⁸ is

as shown below:

For example, Y can include1-palmitoyl-2-oleo-sn-glycero-3-phosphocholine, as shown below:

In some embodiments, Y comprises a structure of Formula D, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; R⁷ ischoline; and R⁸ is acetyl, as shown below:

In some embodiments, Y comprises a structure of Formula D, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; R⁷ ischoline; and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

and derivatives thereof.

In some embodiments, Y comprises a structure of Formula D, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; R⁷ isethanolamine, and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

In some embodiments, Y comprises a structure of Formula D, wherein n is4-26; each R⁵, when present, is independently hydrogen or halogen; R⁷ isinositol, and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula D, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula D that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula D and means of conjugation of Formula D to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula D is conjugated to L or Q through a terminalcarboxylic acid, amino, or hydroxyl moiety.

Grifolic Acid or Derivative Thereof

In some embodiments of the invention, Y is grifolic acid or derivativethereof, as shown in Formula E:

wherein each R⁹ is independently a moiety that permits or promotesagonist or antagonist activity upon binding of the compound of Formula Eto a GPCR. In some embodiments, Y comprises a structure of Formula E,wherein each R⁹ is independently hydrogen, OH, or O(C₁-C₈ alkyl).Nonlimiting examples of Y in these embodiments include grifolic acid andgrifolic acid methyl ether, shown below:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula E, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula E that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula E and means of conjugation of Formula E to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula E is conjugated to L or Q at any position onFormula E. In some embodiments, Formula E is conjugated to L or Qthrough a carboxylic acid or hydroxyl moiety.

Diacetylphloroglucinol or Derivative Thereof

In some embodiments of the invention, Y is diacetylphloroglucinol orderivative thereof, as shown in Formula F:

wherein each m is independently 0-15, and each R¹⁰ is independently amoiety that permits or promotes agonist or antagonist activity uponbinding of the compound of Formula F to a GPCR. In some embodiments, Ycomprises a structure of Formula F, wherein each m is independently0-15; and each R¹⁰ is independently hydrogen or C₁-C₁₈ alkyl. In someembodiments, Y is a compound of Formula F, wherein each m is 9 and eachR¹⁰ is hydrogen, as shown below:

In embodiments wherein Y comprises a structure of Formula F, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula F that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula F and means of conjugation of Formula F to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula F is conjugated to L or Q at any position onFormula F. In some embodiments, Formula F is conjugated to L or Qthrough a hydroxyl moiety.

Retinoic Acid or Derivative Thereof

In some embodiments, Y is retinoic acid or a derivative thereof, asshown in Formula G:

wherein R¹¹ is a moiety that permits or promotes agonist or antagonistactivity upon the binding of the compound of Formula G to a GPCR, and

represents either E or Z stereochemistry. In some embodiments, Ycomprises a structure of Formula G wherein R¹¹ is C(O)OH, CH₂OH, orC(O)H. In some embodiments, Y comprises a structure of Formula G whereinR¹¹ is a carboxylic acid derivative. Nonlimiting examples of thecompound of Formula G include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula G, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula G that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y. Insome embodiments, Formula G is conjugated to L or Q at R¹¹.

Thiazolidinedione Derivative

In some exemplary embodiments, Y is a thiazolidinedione compoundcomprising a structure as described by Formula H:

Nonlimiting examples of the compound of Formula H include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula H, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula H that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula H and means of conjugation of Formula H to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula H is conjugated to L or Q through a hydroxylmoiety, through an aromatic moiety, or through a carboxylic acid ofFormula H.

GPCR Ligands of Low Molecular Weight

In some embodiments of the invention, Y is small molecule having amolecular weight of up to about 5000 daltons, or up to about 2000daltons, or up to about 1000 daltons, or up to about 500 daltons thatpermits or promotes agonist or antagonist activity at a GPCR. In someembodiments, Y is 1000 daltons or less. Nonlimiting examples of smallmolecules that permit or promote agonist or antagonist activity at aGPCR include:

In the embodiments wherein Y is a GPCR ligand of low molecular weight, Yis conjugated to L (e.g. when L is a linking group) or Q (e.g. when L isa bond) at any position of Y that is capable of reacting with Q or L.One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y, suchas, for example, a carboxylic acid or an alcohol moiety.

4-Phenoxymethylpiperidines

In some embodiments of the invention, Y is a 4-phenoxymethylpiperidineor derivative thereof, as shown in Formula J:

wherein R¹⁴ and R¹⁵ are each moieties that permits or promotes agonistor antagonist activity at a GPCR. In some embodiments, Y comprises astructure of Formula J wherein

R¹⁴ is phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl,pyrazol-4-yl, 1H-pyrazol-4-yl, 6-oxo-1,6-dihydropyridin-3-yl,2-oxo-1,2-dihydropyrimidin-5-yl, 2-oxo-1,2-dihydropyridin-4-yl and2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl;

wherein said phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,pyrimidin-5-yl, pyrazol-4-yl, 1H-pyrazol-4-yl,6-oxo-1,6-dihydropyridin-3-yl, 2-oxo-1,2-dihydropyrimidin-5-yl,2-oxo-1,2-dihydropyridin-4-yl or2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl of R¹⁴ is optionallysubstituted with 1 to 3 radicals independently selected from halo,—X¹R¹⁶, —X¹OR¹⁶, —X¹C(O)R¹⁶, —X¹C(O)OR¹⁶, —X²NR¹⁶C(O)R¹⁶, —X¹S(O)₂R¹⁶,—X¹NR¹⁶S(O)₂R¹⁶, —X¹C(O)NR¹⁶R¹⁷, —X¹C(O)NR¹⁶X²OR¹⁷,—X¹C(O)NR¹⁶X²NR¹⁶R¹⁷, —X¹C(O)NR¹⁶X²C(O)OR¹⁷, —X¹S(O)O₂X²R¹⁶,—X¹S(O)₀₋₂X²OR¹⁶, —X²CN, —X¹OX²R¹⁶, —X¹NR¹⁷X²R¹⁶, —X²NR¹⁶R¹⁷,—X¹S(O)₀₋₂X²C(O)R¹⁶, —X¹S(O)₀₋₂X²C(O)OR¹⁶ and —X¹S(O)₀₋₂NR¹⁶R¹⁷;

wherein X¹ is selected from a bond, O, NR¹⁷ and C₁₋₄alkyl; R¹⁷ isselected from hydrogen and C₁₋₆alkyl;

-   -   each X² is independently selected from a bond and C₁₋₄alkyl;    -   each R¹⁶ is independently selected from hydrogen, C₁₋₆alkyl,        C₆₋₁₀aryl, C₁₋₁₀heteroaryl, C₃₋₈heterocycloalkyl, C₃₋₈cycloalkyl        and —X³C(O)OR¹⁸, —X³R¹⁸, —X³OR¹⁸, —X³NR¹⁸R¹⁹;

wherein said alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl ofR¹⁶ is optionally substituted with 1 to 3 radicals independentlyselected from hydroxy, halo, amino, cyano, C₁₋₆alkyl,halo-substituted-C₁₋₆alkyl, hydroxy-substituted-C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkoxy, C₆₋₁₀aryl-C₁₋₄alkoxy and —NR¹⁸C(O)R¹⁹; X³is C₁₋₃alkyl; and

R¹⁸ is selected from hydrogen, C₁₋₆alkyl and C₃₋₈heterocycloalkyloptionally substituted with C₁₋₆alkyl;

R¹⁹ are independently selected from hydrogen and C₁₋₆alkyl;

and R¹⁵ is selected from R²⁰ and —C(O)OR²⁰;

wherein R²⁰ is selected from C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₁₀heteroaryl,C₃₋₈cycloalkyl and C₃₋₈heterocycloalkyl;

wherein said alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl ofR²⁰ is optionally substituted with 1 to 3 radicals independentlyselected from halo, C₁₋₆alkyl, C₃₋₁₂cycloalkyl, C₃₋₈heterocycloalkyl,halo-substituted-C₁₋₆alkyl, hydroxy-substituted-C₁₋₆alkyl, andhalo-substituted-C₁₋₆alkoxy; or the pharmaceutically acceptable saltsthereof.

Nonlimiting examples of 4-phenoxymethylpiperidine compounds that permitor promote agonist or antagonist activity at a GPCR include:2-(4-((2,6-difluoro-4-(5-(methylsulfonyl)pyridin-2-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;tert-butyl4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidine-1-carboxylate;2-(4-((3,5-difluoro-4′-(3-methyloxetan-3-yl)methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4((3,5-difluoro-4′-(isobutylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(propylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(isopropylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-carbonitrile;4′4(1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-carbonitrile;2(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-yl)acetonitrile;(4-(((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-yl)methanol;2-(4-((3,5-difluoro-3′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)acetonitrile;(4′-(4(1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methanol;2-(4-((2,6-difluoro-4-(2-(pyrrolidin-1-yl)pyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(pyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(pyridin-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;tert-butyl4-((2,6-difluoro-4-(2-methoxypyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;tert-butyl4-((2,6-difluoro-4-(pyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;2-(4-((2,6-difluoro-4-(6-(methylsulfonyl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4-(6-chloro-2-methylpyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(1-methyl-1H-pyrazol-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(1H-pyrazol-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)picolinonitrile;2-(4-((2,6-difluoro-4-(3-methoxypyridin-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;N-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)acetamide;N-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methanesulfonamide;2-(4-((4-(6-(benzyloxy)pyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridin-2(1H)-one;2-(4-((2,6-difluoro-4-(2-methoxypyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidin-2(1H)-one;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidine-2-carboxamide;tert-butyl4-((2,6-difluoro-4-(pyridin-3-yl)phenoxy)methyl)piperidine-1-carboxylate;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidine-2-carboxylicacid;2-(5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridin-2-yl)propan-2-ol;2-(4-((3,5-difluoro-4′-(1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3′-((2H-tetrazol-5-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-((2H-tetrazol-5-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4-(6-(2H-tetrazol-5-yl)pyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(2-methyl-2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(1-methyl-1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(2-methyl-2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(1-methyl-1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-((2-methyl-2H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-((1-methyl-1H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-((2-methyl-2H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-((1-methyl-1H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(6-(1-methyl-1H-tetrazol-5-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;1-methylcyclopropyl4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidine-1-carboxylate;4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridine1-oxide; tert-butyl4-((2,6-difluoro-4-(2-oxo-1,2-dihydropyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;tert-butyl4-((2,6-difluoro-4-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;2-(4-((2,6-difluoro-4-(2-methyl-6-(1H-1,2,4-triazol-1-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(2-methyl-6-(methylsulfonyl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)oxazole;2-(4-((3,5-difluoro-4′-(1H-pyrazol-3-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;1-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)-N-methylmethanamine;N-((4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methyl)-2-methylpropan-2-amine;4-((4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methyl)morpholino;2-(4-((4′-((1H-imidazol-1-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-(2H-tetrazol-2-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-((1H-tetrazol-1-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)picolinamide;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)-N-methylpicolinamide;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)-N,N-dimethylpicolinamide;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(piperazine-1-carbonyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((4-(2-(dimethylcarbamoyl)pyrimidin-5-yl)-2,6-difluorophenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(morpholine-4-carbonyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(2-methoxyethylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(2-hydroxyethylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;and 1-methylcyclopropyl4-((2,6-difluoro-4-(2-(3-hydroxypropylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate,or any of the compounds disclosed in WO 2010/006191, which isincorporated herein by reference in its entirety.

In embodiments wherein Y comprises a structure of Formula J, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula J that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula J and means of conjugation of Formula J to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula J is conjugated to L or Q at any of position onFormula J.

GPCR Ligand that Acts at GPR30

In some embodiments of the invention, Y acts at GPR30. In someembodiments, Y can have any structure that permits or promotes agonistactivity upon binding to GPR30, while in other embodiments Y is anantagonist of GPR30. In some embodiments, Y acts at GPR30 and isestrogen or a derivative of estrogen, as can be shown by Formula I:

wherein R¹, R¹² and R¹³ are moieties that permit or promote agonist orantagonist activity upon binding of the compound of Formula I to GPR30.In some embodiments, Formula I further comprises one or moresubstitutents at one or more of positions 1, 2, 4, 6, 7, 8, 9, 11, 12,14, 15, and 16 (e.g. a ketone at position-6).

In some embodiments when Y comprises a structure of Formula I, wherein

R¹ is hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl,heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl,(C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈ alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)OH, C₀-C₈ alkyl)C(O)O aryl, (C₀-C₈ alkyl)C(O)O heteroaryl,(C₀-C₈ alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, or SO₃H;

R¹² is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₁₈alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀-C₈ alkyl)NR²⁴C₁-C₁₈alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₁₈ alkynyl,(C₀-C₈ alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl,(C₀-C₈ alkyl)C(O)O heteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)OC(O)C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈alkyl)NR²⁴C(O)C₁-C₁₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈alkyl)NR²⁴C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈alkyl)OC(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)OC(O)OC₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)OH, (C₀-C₈alkyl)OC(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈alkyl)NR²⁴(O)OC₁-C₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₁₈ alkenyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₁₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(O)OH; (C₀-C₈alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)OH, (C₀-C₈ alkyl)OC(O)C₁-C₁₈ alkyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴C₁-C₁₈alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈ alkyl)NR²⁴C(O)C₁-C₁₈ alkyl,(C₀-C₈ alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈ alkyl)NR²⁴C(O)C₂-C₁₈alkynyl, or (C₀-C₈ alkyl)NR²⁴C(O)OH;

R¹³ is hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl,heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)C(O)C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₁₈ alkenyl, (C₀-C₈alkyl)C(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl,(C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈ alkyl)C(O)OC₁-C₁₈ alkyl, (C₀-C₈alkyl)C(O)OC₂-C₁₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₁₈ alkynyl, (C₀-C₈alkyl)C(O)OH, C₀-C₈ alkyl)C(O)O aryl, (C₀-C₈ alkyl)C(O)O heteroaryl,(C₀-C₈ alkyl)C(O)NR²⁴C₁-C₁₈ alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkenyl,(C₀-C₈ alkyl)C(O)NR²⁴C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈alkyl)C(O)NR²⁴aryl, (C₀-C₈ alkyl)C(O)NR²⁴heteroaryl, or SO₃H; and,

R²⁴ is hydrogen or C₁-C₁₈ alkyl.

In some embodiments, Y acts at GPR30 and comprises a structure ofFormula I, wherein

R¹ is hydrogen, C₁-C₇ alkyl; (C₀-C₃ alkyl)C(O)C₁-C₇ alkyl, (C₀-C₃alkyl)C(O)aryl, or SO₃H;

R¹² is hydrogen, (C₀-C₈ alkyl)halo, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂₋₁₈alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈alkyl)OC₁-C₈ alkyl, (C₀-C₈ alkyl)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₈alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀-C₈ alkyl)NR²⁴C₁-C₈ alkyl,(C₀-C₈ alkyl)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkynyl, (C₀-C₈alkyl)NR²⁴H₂, (C₀-C₈ alkyl)C(O)C₁-C₈ alkyl, (C₀-C₈ alkyl)C(O)C₂-C₈alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(O)H, (C₀-C₈alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl, (C₀-C₈ alkyl)C(O)OC₁-C₈alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)OC₂-C₈ alkynyl,(C₀-C₈ alkyl)C(O)OH, (C₀-C₈ alkyl)C(O)O aryl, (C₀-C₈ alkyl)C(O)Oheteroaryl, (C₀-C₈ alkyl)OC(O)C₁-C₈ alkyl, (C₀-C₈ alkyl)OC(O)C₂-C₈alkenyl, (C₀-C₈ alkyl)OC(O)C₂-C₁₈ alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴C₁-C₈alkyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₈alkynyl, (C₀-C₈ alkyl)C(O)NR²⁴H₂, (C₀-C₈ alkyl)C(O)NR²⁴aryl, (C₀-C₈alkyl)C(O)NR²⁴heteroaryl, (C₀-C₈ alkyl)NR²⁴C(O)C₁-C₈ alkyl, (C₀-C₈alkyl)NR²⁴C(O)C₂-C₈ alkenyl, or (C₀-C₈ alkyl)NR²⁴C(O)C₂-C₈ alkynyl,(C₀-C₈ alkyl)NR²⁴C(O)OH, (C₀-C₈ alkyl)OC(O)OC₁-C₈ alkyl, (C₀-C₈alkyl)OC(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(O)OC₂-C₈ alkynyl, (C₀-C₈alkyl)OC(O)OH, (C₀-C₈ alkyl)OC(O)NR²⁴C₁-C₈ alkyl, (C₀-C₈alkyl)OC(O)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(O)NR²⁴C₂-C₈ alkynyl,(C₀-C₈ alkyl)OC(O)NR²⁴H₂, (C₀-C₈ alkyl)NR²⁴(O)OC₁-C₈ alkyl, (C₀-C₈alkyl)NR²⁴(O)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴(O)OC₂-C₈ alkynyl, or(C₀-C₈ alkyl)NR²⁴(O)OH;

R¹³ is hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, heteroalkyl,(C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈ alkyl)C(O)C₁-C₈alkyl, (C₀-C₈ alkyl)C(O)C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)C₂-C₈ alkynyl,(C₀-C₈ alkyl)C(O)H, (C₀-C₈ alkyl)C(O)aryl, (C₀-C₈ alkyl)C(O)heteroaryl,(C₀-C₈ alkyl)C(O)OC₁-C₈ alkyl, (C₀-C₈ alkyl)C(O)OC₂-C₈ alkenyl, (C₀-C₈alkyl)C(O)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)C(O)OH, C₀-C₈ alkyl)C(O)O aryl,(C₀-C₈ alkyl)C(O)O heteroaryl, (C₀-C₈ alkyl)C(O)NR²⁴C₁-C₈ alkyl, (C₀-C₈alkyl)C(O)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(O)NR²⁴C₂-C₈ alkynyl, (C₀-C₈alkyl)C(O)NR²⁴H₂, (C₀-C₈ alkyl)C(O)NR²⁴aryl, or (C₀-C₈alkyl)C(O)NR²⁴heteroaryl; and

R²⁴ is hydrogen or C₁-C₇ alkyl.

For example, R¹ is hydrogen, propionate, acetate, benzoate, or sulfate;R¹² is hydrogen, ethynyl, hydroxyl; and R¹³ is acetate, cypionate,hemisucciniate, enanthate, or propionate.

Nonlimiting examples of the compound of Formula I include 17β-estradiol,modified forms of estradiol such as β-estradiol 17-acetate, β-estradiol17-cypionate, β-estradiol 17-enanthate, β-estradiol 17-valerate,β-estradiol 3,17-diacetate, β-estradiol 3,17-dipropionate, β-estradiol3-benzoate, β-estradiol 3-benzoate 17-n-butyrate, β-estradiol 3-glycidylether, β-estradiol 3-methyl ether, β-estradiol 6-one, β-estradiol3-glycidyl, β-estradiol 6-one 6-(O-carboxymethyloxime), 16-epiestriol,17-epiestriol, 2-methoxy estradiol, 4-methoxy estradiol, estradiol17-phenylpropionate, and 17β-estradiol 2-methyl ether,17α-ethynylestradiol, megestrol acetate, estriol, and derivativesthereof. In some embodiments, carbon 17 has a ketone substitutent and R⁵and R⁶ are absent (e.g. estrone). Some of the aforementioned compoundsof Formula I are shown below:

Other examples of ligands that act at GPR30 are described in Martenssonet al., Endocrinology 150(2):687-698 (2009), which is incorporatedherein by reference in its entirety for each of its disclosures of GPR30ligands.

In embodiments wherein Y comprises a structure of Formula I, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula I that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula I and means of conjugation of Formula I to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula I is conjugated to L or Q at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofFormula I. In some embodiments, Formula B is conjugated to L or Q atposition 3 or 17 of Formula I.

GPCR Ligand that Acts at GPR40

In some embodiments of the invention, Y acts at GPR40. In someembodiments, Y can have any structure that permits or promotes agonistactivity upon binding to GPR40, while in other embodiments Y is anantagonist of GPR40.

In some embodiments, Y acts at GPR40 and is a free fatty acid orderivative thereof, as shown in Formula B:

wherein n is 0-26 and each R⁵, when present, are each independently amoiety that permits or promotes agonist or antagonist activity uponbinding of the compound of Formula B to GPR40. In some embodiments, Yacts at GPR40 and comprises a structure of Formula B, wherein n is 4-26and each R⁵, when present, are each independently a moiety that permitsor promotes agonist or antagonist activity upon binding of the compoundof Formula B to GPR40. In some embodiments, Y acts at GPR40 andcomprises a structure of Formula B, wherein n is 4-26 and each R⁵, whenpresent, is independently hydrogen, C₁-C₇ alkyl or halogen. Nonlimitingexamples of Y is these embodiments include: n-caproic acid, heptanoicacid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauricacid, tridecanoic acid, myristic acid, pentadeconoic acid, palmiticacid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidicacid, heneicosanoic acid, behenic acid, and tricosanoic acid. In someembodiments Y acts at GPR40 and is unsaturated with either cis or transstereochemistry such as, for example, mead acid, myristoleic acid,palmitoleic acid, sapienic acid, oleic acid, linoleic acid, α-linolenicacid, elaidic acid, retinal, tretinoin, petroselinic acid, arachidonicacid, dihydroxyeicosatetraenoic acid (DiHETE), octadecynoic acid,eicosatriynoic acid, eicosadienoic acid, eicosatrienoic acid,eicosapentaenoic acid, erucic acid, dihomolinolenic acid, docosatrienoicacid, docosapentaenoic acid, docosahexaenoic acid, and adrenic acid. Inexemplary embodiments, Y acts at GPR40 and is pentanoic acid, palmiticacid, linoleic acid, or eicosatrienoic acid.

In some embodiments, Y acts at GPR40 and comprises a structure ofFormula B further modified with a moiety (e.g. carboxylic acid,carboxylic acid derivative, amide) at its tail. In some embodiments, Yacts at GPR40 and is MEDICA16, as shown below:

In embodiments wherein Y comprises a structure of Formula B, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula B that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula B and means of conjugation of Formula B to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula B is conjugated to L or Q at any position onFormula B. In some embodiments, Formula B is conjugated to L or Qthrough the terminal carboxylic acid moiety.

In some embodiments, Y acts at GPR40 and is diacetylphloroglucinol or aderivative thereof, as shown in Formula F:

wherein each m is independently 0-15, and each R¹⁰ is independently amoiety that permits or promotes agonist or antagonist activity uponbinding of the compound of Formula F to GPR40. In some embodiments, Yacts at GPR40 and comprises a structure of Formula F, wherein each m isindependently 0-15; and each R¹⁰ is independently hydrogen or C₁-C₁₈alkyl. In some embodiments, Y acts at GPR40 and is a compound of FormulaF, wherein each m is 9 and each R¹⁰ is hydrogen, as shown below:

In embodiments wherein Y comprises a structure of Formula F, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula F that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula F and means of conjugation of Formula F to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula F is conjugated to L or Q at any position onFormula F. In some embodiments, Formula F is conjugated to L or Qthrough a hydroxyl moiety.

In some embodiments, Y acts at GPR40 and is retinoic acid or aderivative thereof, as shown in Formula G:

wherein R¹¹ is a moiety that permits or promotes agonist or antagonistactivity upon the binding of the compound of Formula G to GPR40, and

represents either E or Z stereochemistry. In some embodiments, Y acts atGPR40 and comprises a structure of Formula G wherein R¹¹ is C(O)OH,CH₂OH, or C(O)H. In some embodiments, Y acts at GPR40 and comprises astructure of Formula G wherein R¹¹ is a carboxylic acid derivative.Nonlimiting examples of the compound of Formula G include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula G, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula G that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y. Insome embodiments, Formula G is conjugated to L or Q at R¹¹.

In some embodiments, Y acts at GPR40 and is a thiazolidinedione compoundcomprising a structure as described by Formula H:

Nonlimiting examples of the compound of Formula H include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula H, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula H that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula H and means of conjugation of Formula H to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula H is conjugated to L or Q at any position onFormula H.

In some embodiments, Y is a small molecule having a molecular weight ofup to about 5000 daltons, or up to about 2000 daltons, or up to about1000 daltons, or up to about 500 daltons that permits or promotesagonist or antagonist activity at GPR40. In some embodiments, Y is 1000daltons or less. Nonlimiting examples of small molecules that permit orpromote agonist or antagonist activity at GPR40 include:

In the embodiments wherein Y is a GPCR ligand of low molecular weight, Yis conjugated to L (e.g. when L is a linking group) or Q (e.g. when L isa bond) at any position of Y that is capable of reacting with Q or L.One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Formula H is conjugated to L or Q through a hydroxylmoiety, through an aromatic moiety, or through a carboxylic acid ofFormula H.

In some embodiments, Y is grifolic acid or a derivative thereof, whichpermits or promotes antagonist activity at GPR40, as shown in Formula E:

wherein each R⁹ is independently a moiety that permits or promotesantagonist activity upon binding of the compound of Formula E to GPR40.In some embodiments, Y acts at GPR40 and comprises a structure ofFormula E, wherein each R⁹ is independently hydrogen, OH, or O(C₁-C₈alkyl). Nonlimiting examples of Y in these embodiments include grifolicacid and grifolic acid methyl ether, shown below:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula E, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula E that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula E and means of conjugation of Formula E to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula E is conjugated to L or Q at any position onFormula E. In some embodiments, Formula E is conjugated to L or Qthrough a carboxylic acid or hydroxyl moiety.

Other examples of ligands that act at GPR40 are described in Briscoe etal., J. Biol. Chem., 278:11303-11311 (2003); Briscoe et al., Br. J.Pharmacol. 148:619-628 (2006); Davi et al., Chem. Commun. 40:4974-4976(2008); Kebede et al., Diabetes, 57:2432-2437 (2008); Hara et al., Mol.Pharmacol. 75:85-91 (2009); Hirasawa et al., Biol. Pharm. Bull.31:1847-1851 (2008); Suzuki et al., J. Med. Chem. 51:7640-7644 (2008);Tikhonove et al., J. Med. Chem. 50:2981-2989 (2007), and Zhou et al.Bioorg. Med. Chem. Lett. 18:6357-6361 (2008); each and all of which areincorporated herein by reference for each of their disclosures of GPR40ligands.

GPCR Ligand that Acts on GPR119

In some embodiments of the invention, Y acts at GPR119. In someembodiments, Y can have any structure that permits or promotes agonistactivity upon binding to GPR119, while in other embodiments Y is anantagonist of GPR119.

In some embodiments, Y acts at GPR119 and is a free fatty amide orderivative thereof, as shown in Formula C:

wherein n is 0-26 and each R⁵, when present, is independently a moietythat permits or promotes agonist or antagonist activity upon binding ofthe compound of Formula C to GPR119. In some embodiments, Y acts atGPR119 and comprises a structure of Formula C, wherein n is 0-26; eachR⁵, when present, is independently hydrogen, C₁-C₇ alkyl, or halogen;and R⁶ is hydrogen, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)NH₂,(C₀-C₈ alkyl)halo, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, heteroalkyl, (C₀-C₈alkyl)aryl, or (C₀-C₈ alkyl)heteroaryl. In some embodiments Formula C issaturated, while is other embodiments, Formula C is unsaturated witheither cis or trans stereochemistry.

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula C, wherein n is 4-26; each R⁵, when present, is hydrogen; and R⁶is hydrogen. For example, Y acts at GPR119 and is oleamide, as shownbelow:

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula C, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; and R⁶ is (C₁-C₈ alkyl)OH. Nonlimiting examples ofcompounds of Formula C when R⁶ is (C₁-C₈ alkyl)OH include:

and derivatives thereof.

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula C, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; and R⁶ is (C₁-C₃ alkyl)aryl. For example, Y acts atGPR119 and is N-oleoyldopamine, as shown below:

In embodiments wherein Y comprises a structure of Formula C, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula C that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula C and means of conjugation of Formula C to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula C is conjugated to L or Q at any position onFormula C. In some embodiments, Formula C is conjugated to L or Qthrough a terminal carboxylic acid or hydroxyl moiety.

In some embodiments of the invention, Y acts at GPR119 and is aphospholipid or derivative thereof, as shown in Formula D:

wherein n is 0-26, each R⁵, R⁷, and R⁸ are moieties that permit orpromote agonist or antagonist activity upon binding of the compound ofFormula D to GPR119. In some embodiments, Y acts at GPR119 and comprisesa structure of Formula D, wherein n is 0-26; each R⁵, when present, isindependently hydrogen, C₁-C₇ alkyl, or halogen; R⁷ is choline,ethanolamine, or inositol; and R⁸ is hydrogen or

In some embodiments Formula D is saturated, while in other embodiments,Formula D is unsaturated with either cis or trans stereochemistry.

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula D, wherein each n is independently 4-26; each R⁵, when present,is independently hydrogen or halogen; R⁷ is choline; and R⁸ is

as shown below:

For example, Y acts at GPR119 and is1-palmitoyl-2-oleo-sn-glycero-3-phosphocholine, as shown below:

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula D, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; R⁷ is choline; and R⁸ is acetyl, as shown below:

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula D, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; R⁷ is choline; and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

and derivatives thereof.

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula D, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; R⁷ is ethanolamine, and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

In some embodiments, Y acts at GPR119 and comprises a structure ofFormula D, wherein n is 4-26; each R⁵, when present, is independentlyhydrogen or halogen; R⁷ is inositol, and R⁸ is OH, as shown below:

Nonlimiting examples of Y in these embodiments include:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula D, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula D that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula D and means of conjugation of Formula D to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula D is conjugated to L or Q through a terminalcarboxylic acid, amino, or hydroxyl moiety.

In some embodiments, Y acts at GPR119 and is retinoic acid or aderivative thereof, as shown in Formula G:

wherein R¹¹ is a moiety that permits or promotes agonist or antagonistactivity upon the binding of the compound of Formula G to GPR119, and

represents either E or Z stereochemistry. In some embodiments, Y acts atGPR119 and comprises a structure of Formula G wherein R¹¹ is C(O)OH,CH₂OH, or C(O)H. In some embodiments, Y acts at GPR119 and comprises astructure of Formula G wherein R¹¹ is a carboxylic acid derivative.Nonlimiting examples of the compound of Formula G include:

and derivatives thereof. In some embodiments, Y acts at GP119 and isall-trans-retinoic acid.

In embodiments wherein Y comprises a structure of Formula G, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula G that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y. Insome embodiments, Formula G is conjugated to L or Q at R¹¹.

In some embodiments of the invention, Y is small molecule having amolecular weight of up to about 5000 daltons, or up to about 2000daltons, or up to about 1000 daltons, or up to about 500 daltons thatpermits or promotes agonist or antagonist activity at GPR119. In someembodiments, Y is 1000 daltons or less. Nonlimiting examples of smallmolecules that permit or promote agonist or antagonist activity at aGPR119 include:

and derivatives thereof.

In the embodiments wherein Y is a GPCR ligand of low molecular weight, Yis conjugated to L (e.g. when L is a linking group) or Q (e.g. when L isa bond) at any position of Y that is capable of reacting with Q or L.One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y, suchas, for example, a carboxylic acid or an alcohol moiety.

4-Phenoxymethylpiperidines

In some embodiments of the invention, Y acts at GPR119 and comprises astructure of Formula J:

wherein R¹⁴ and R¹⁵ are each moieties that permits or promotes agonistor antagonist activity at a GPCR. In some embodiments, Y comprises astructure of Formula J wherein

R¹⁴ is phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl,pyrazol-4-yl, 1H-pyrazol-4-yl, 6-oxo-1,6-dihydropyridin-3-yl,2-oxo-1,2-dihydropyrimidin-5-yl, 2-oxo-1,2-dihydropyridin-4-yl and2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl;

wherein said phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,pyrimidin-5-yl, pyrazol-4-yl, 1H-pyrazol-4-yl,6-oxo-1,6-dihydropyridin-3-yl, 2-oxo-1,2-dihydropyrimidin-5-yl,2-oxo-1,2-dihydropyridin-4-yl or2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl of R¹⁴ is optionallysubstituted with 1 to 3 radicals independently selected from halo,—X¹R¹⁶, —X¹OR¹⁶, —X¹C(O)R¹⁶, —X¹C(O)OR¹⁶, —X²NR¹⁶C(O)R¹⁶, —X¹S(O)₂R¹⁶,—X¹NR¹⁶S(O)₂R¹⁶, —X¹C(O)NR¹⁶R¹⁷, —X¹C(O)NR¹⁶X²OR¹⁷,—X¹C(O)NR¹⁶X²NR¹⁶R¹⁷, —X¹C(O)NR¹⁶X²C(O)OR¹⁷, —X¹S(O)O₂X²R¹⁶,—X¹S(O)₀₋₂X²OR¹⁶, —X²CN, —X¹OX²R¹⁶, —X¹NR¹⁷X²R¹⁶, —X²NR¹⁶R¹⁷,—X¹S(O)₀₋₂X²C(O)R¹⁶, —X¹S(O)₀₋₂X²C(O)OR¹⁶ and —X¹S(O)₀₋₂NR¹⁶R¹⁷;

wherein X¹ is selected from a bond, O, NR¹⁷ and C₁₋₄alkyl;

R¹⁷ is selected from hydrogen and C₁₋₆alkyl;

each X² is independently selected from a bond and C₁₋₄alkyl;

each R¹⁶ is independently selected from hydrogen, C₁₋₆alkyl, C₆₋₁₀aryl,C₁₋₁₀heteroaryl, C₃₋₈heterocycloalkyl, C₃₋₈cycloalkyl and —X³C(O)OR¹⁸,—X³R¹⁸, —X³OR¹⁸, —X³NR¹⁸R¹⁹;

wherein said alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl ofR¹⁶ is optionally substituted with 1 to 3 radicals independentlyselected from hydroxy, halo, amino, cyano, C₁₋₆alkyl,halo-substituted-C₁₋₆alkyl, hydroxy-substituted-C₁₋₆alkyl, C₁₋₆alkoxy,halo-substituted-C₁₋₆alkoxy, C₆₋₁₀aryl-C₁₋₄alkoxy and —NR¹⁸C(O)R¹⁹; X³is C₁₋₃alkyl; and

R¹⁸ is selected from hydrogen, C₁₋₆alkyl and C₃₋₈heterocycloalkyloptionally substituted with C₁₋₆alkyl;

R¹⁹ are independently selected from hydrogen and C₁₋₆alkyl;

and R¹⁵ is selected from R²⁰ and —C(O)OR²⁰;

wherein R²⁰ is selected from C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₁₀heteroaryl,C₃₋₈cycloalkyl and C₃₋₈heterocycloalkyl;

wherein said alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl ofR²⁰ is optionally substituted with 1 to 3 radicals independentlyselected from halo, C₁₋₆alkyl, C₃₋₁₂cycloalkyl, C₃₋₈heterocycloalkyl,halo-substituted-C₁₋₆alkyl, hydroxy-substituted-C₁₋₆alkyl, andhalo-substituted-C₁₋₆alkoxy; or the pharmaceutically acceptable saltsthereof.

Nonlimiting examples of 4-phenoxymethylpiperidine compounds that permitor promote agonist or antagonist activity at a GPCR include:2-(4-((2,6-difluoro-4-(5-(methylsulfonyl)pyridin-2-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;tert-butyl4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidine-1-carboxylate;2-(4-((3,5-difluoro-4′-(3-methyloxetan-3-yl)methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4((3,5-difluoro-4′-(isobutylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(propylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(44(3,5-difluoro-4′-(isopropylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-carbonitrile;4′4(1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-carbonitrile;2-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-yl)acetonitrile;(4′4(1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-3-yl)methanol;2-(4-((3,5-difluoro-3′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)acetonitrile;(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methanol;2-(4-((2,6-difluoro-4-(2-(pyrrolidin-1-yl)pyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(pyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(pyridin-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;tert-butyl4-((2,6-difluoro-4-(2-methoxypyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;tert-butyl4-((2,6-difluoro-4-(pyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;2-(4-((2,6-difluoro-4-(6-(methylsulfonyl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4-(6-chloro-2-methylpyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(1-methyl-1H-pyrazol-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(1H-pyrazol-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)picolinonitrile;2-(4-((2,6-difluoro-4-(3-methoxypyridin-4-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;N-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)acetamide;N-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methanesulfonamide;2-(4-((4-(6-(benzyloxy)pyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridin-2(1H)-one;2-(4-((2,6-difluoro-4-(2-methoxypyrimidin-5-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidin-2(1H)-one;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidine-2-carboxamide;tert-butyl4-((2,6-difluoro-4-(pyridin-3-yl)phenoxy)methyl)piperidine-1-carboxylate;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyrimidine-2-carboxylicacid;2-(5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridin-2-yl)propan-2-ol;2-(4-((3,5-difluoro-4′-(1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3′-((2H-tetrazol-5-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-((2H-tetrazol-5-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4-(6-(2H-tetrazol-5-yl)pyridin-3-yl)-2,6-difluorophenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(2-methyl-2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-(1-methyl-1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(2-methyl-2H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-(1-methyl-1H-tetrazol-5-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-((2-methyl-2H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-3′-((1-methyl-1H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-((2-methyl-2H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((3,5-difluoro-4′-((1-methyl-1H-tetrazol-5-yl)methyl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(6-(1-methyl-1H-tetrazol-5-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;1-methylcyclopropyl4-((3,5-difluoro-4′-(methylsulfonyl)biphenyl-4-yloxy)methyl)piperidine-1-carboxylate;4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)pyridine1-oxide; tert-butyl4-((2,6-difluoro-4-(2-oxo-1,2-dihydropyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;tert-butyl4-((2,6-difluoro-4-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)phenoxy)methyl)piperidine-1-carboxylate;2-(4-((2,6-difluoro-4-(2-methyl-6-(1H-1,2,4-triazol-1-yl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((2,6-difluoro-4-(2-methyl-6-(methylsulfonyl)pyridin-3-yl)phenoxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)oxazole;2-(4-((3,5-difluoro-4′-(1H-pyrazol-3-yl)biphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;1-(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)-N-methylmethanamine;N-((4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methyl)-2-methylpropan-2-amine;44(4′-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3′,5′-difluorobiphenyl-4-yl)methyl)morpholino;2-(4-((4′-((1H-imidazol-1-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-(2H-tetrazol-2-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;2-(4-((4′-((1H-tetrazol-1-yl)methyl)-3,5-difluorobiphenyl-4-yloxy)methyl)piperidin-1-yl)-5-ethylpyrimidine;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)picolinamide;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)-N-methylpicolinamide;5-(4-((1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)methoxy)-3,5-difluorophenyl)-N,N-dimethylpicolinamide;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(piperazine-1-carbonyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((4-(2-(dimethylcarbamoyl)pyrimidin-5-yl)-2,6-difluorophenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(morpholine-4-carbonyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(2-methoxyethylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl)phenoxy)methyl)piperidine-1-carboxylate;1-methylcyclopropyl4-((2,6-difluoro-4-(2-(2-hydroxyethylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate;and 1-methylcyclopropyl4-((2,6-difluoro-4-(2-(3-hydroxypropylcarbamoyl)pyrimidin-5-yl)phenoxy)methyl)piperidine-1-carboxylate,or any of the compounds disclosed in WO 2010/006191, which isincorporated herein by reference in its entirety.

In embodiments wherein Y comprises a structure of Formula J, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula J that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula J and means of conjugation of Formula J to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula J is conjugated to L or Q at any of position onFormula J.

Other examples of ligands that act at GPR119 are described in Overton etal., Cell Metabolism 3:167-175 (2006); Overton et al., British Journalof Pharmacology 153:S76-S81 (2008), and WO 2010/006191, each and all ofwhich are incorporated herein by reference for each of its disclosuresof GPR119 ligands.

GPCR Ligand that Acts on GPR120

In some embodiments of the invention, Y acts at GPR120. In someembodiments, Y can have any structure that permits or promotes agonistactivity upon binding to GPR120, while in other embodiments Y is anantagonist of GPR120.

In some embodiments, Y acts at GPR120 and is an unsaturated free fattyacid or derivative thereof with cis or trans stereochemistry, as shownin Formula B:

wherein n is 4-26 and each R⁵, when present, are each independently amoiety that permits or promotes agonist or antagonist activity uponbinding of the compound of Formula B to GPR120. In some embodiments, Yacts at GPR120 and comprises a structure of Formula B, wherein n is 4-26and each R⁵, when present, is independently hydrogen, C₁-C₇ alkyl orhalogen. Nonlimiting examples of Y is these embodiments include: meadacid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid,linoleic acid, α-linolenic acid, elaidic acid, petroselinic acid,arachidonic acid, dihydroxyeicosatetraenoic acid (DiHETE), octadecynoicacid, eicosatriynoic acid, eicosadienoic acid, eicosatrienoic acid,eicosapentaenoic acid, erucic acid, dihomolinolenic acid, docosatrienoicacid, docosapentaenoic acid, docosahexaenoic acid, and adrenic acid.

In embodiments wherein Y comprises a structure of Formula B, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula B that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula B and means of conjugation of Formula B to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula B is conjugated to L or Q at any position onFormula B. In some embodiments, Formula B is conjugated to L or Qthrough the terminal carboxylic acid moiety.

In some embodiments, Y acts at GPR120 and is grifolic acid or aderivative thereof, as shown in Formula E:

wherein each R⁹ is independently a functional group that permits orpromotes agonist or antagonist activity upon binding of the compound ofFormula E to GPR120. In some embodiments, Y acts at GPR120 and comprisesa structure of Formula E, wherein each R⁹ is independently hydrogen, OH,or O(C₁-C₈ alkyl). Nonlimiting examples of Y in these embodimentsinclude grifolic acid and grifolic acid methyl ether, shown below:

and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula E, Y isconjugated to L (e.g. when L is a linking group) or Q (e.g. when L is abond) at any position of Formula E that is capable of reacting with Q orL. One skilled in the art could readily determine the position ofconjugation on Formula E and means of conjugation of Formula E to Q or Lin view of general knowledge and the disclosure provided herein. In someembodiments, Formula E is conjugated to L or Q at any position onFormula E. In some embodiments, Formula E is conjugated to L or Qthrough a carboxylic acid or hydroxyl moiety.

In some embodiments, Y is a small molecule having a molecular weight ofup to about 5000 daltons, or up to about 2000 daltons, or up to about1000 daltons, or up to about 500 daltons that permits or promotesagonist or antagonist activity at GPR120. In some embodiments, Y is 1000daltons or less. For example Y acts at GPR120 and is GW9508, as shownbelow:

In the embodiments wherein Y is a GPCR ligand of low molecular weight, Yis conjugated to L (e.g. when L is a linking group) or Q (e.g. when L isa bond) at any position of Y that is capable of reacting with Q or L.One skilled in the art could readily determine the position ofconjugation on Y and means of conjugation of Y to Q or L in view ofgeneral knowledge and the disclosure provided herein. In someembodiments, Y is conjugated to L or Q through any position of Y.

Other examples of ligands that act at GPR120 are described in; Briscoeet al., Br. J. Pharmacol. 148:619-628 (2006); Hara et al., Mol.Pharmacol. 75:85-91 (2009); and Hirasawa et al., Biol. Pharm. Bull.31:1847-1851 (2008), each and all of which are incorporated herein byreference for each of their disclosures of GPR120 ligands.

Modification of the GPCR Ligand (Y)

In some embodiments, the GPCR ligand is derivatized or otherwisechemically modified to comprise a reactive functional group that iscapable of reacting with the glucagon superfamily peptide (Q) or thelinking group (L). In the embodiments described herein, Y is derivatizedat any position of Y that is capable of reacting with Q or L. Theposition of derivatization on Y is apparent to one skilled in the artand depends on the type of GPCR ligand used and the activity that isdesired. In some embodiments, Y is derivatized at any of positions 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25. Other positions of derivatization can be as previouslydescribed herein

The GPCR ligand can be derivatized using any agent known to one skilledin the art or described herein (e.g. see The Linking Group section andthe Chemical Modification of Q and/or Y subsection). For example,estradiol can be derivatized with succinic acid, succinic anhydride,benzoic acid, ethyl 2-bromoacetate, or iodoacetic acid to form the belowderivatives of estradiol that are capable of conjugating to Q or L.

Similarly, any of the aforementioned GPCR ligands can be derivatized bymethods known in the art. Additionally, certain derivatized ligands arecommercially available and can be purchased from chemical companies suchas Sigma-Aldrich.

The Glucagon Superfamily Peptide (O)

In the Q-L-Y conjugates described herein, Q is a glucagon superfamilypeptide. A glucagon superfamily peptide refers to a group of peptidesrelated in structure in their N-terminal and/or C-terminal regions (see,for example, Sherwood et al., Endocrine Reviews 21: 619-670 (2000)). Itis believed that the C-terminus generally functions in receptor bindingand the N-terminus generally functions in receptor signaling. A fewamino acids in the N-terminal and C-terminal regions are highlyconserved among members of the glucagon superfamily. Some of theseconserved amino acids include Hist, Gly4, Phe6, Phe22, Val23, Trp25 andLeu26, with amino acids at these positions showing identity,conservative substitutions or similarity in the structure of their aminoacid side chains.

Glucagon superfamily peptides include glucagon related peptides, GrowthHormone Releasing Hormone (GHRH; SEQ ID NO: 1619), vasoactive intestinalpeptide (VIP; SEQ ID NO: 1620), pituitary adenylate cyclase-activatingpolypeptide 27 (PACAP-27; SEQ ID NO: 1621), peptide histidine isoleucine(PHI; SEQ ID NO: 1542), peptide histidine methionine (PHM; SEQ ID NO:1622), secretin (SEQ ID NO: 1623), and/or analogs, derivatives orconjugates thereof. In some embodiments, Q comprises an amino acidsequence of native glucagon, native exendin-4, native GLP-1(7-37),native GLP-2, native GHRH, native VIP, native PACAP-27, native PHM,native oxyntomodulin, native secretin, or native GIP with up to 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

In some aspects of this invention, Q is a glucagon related peptide suchas, for example, glucagon (SEQ ID NO: 1601), oxyntomodulin (SEQ ID NO:1606), exendin-4 (SEQ ID NO: 1618), glucagon-like peptide-1 (GLP-1)(amino acids 7-36 provided as SEQ ID NO: 1603; amino acids 7-37 areprovided as SEQ ID NO: 1604), glucagon-like peptide-2 (GLP-2, SEQ ID NO:1608), gastric inhibitory peptide (GIP, SEQ ID NO: 1607) or analogs,derivatives and conjugates thereof. A glucagon related peptide hasbiological activity (as an agonist or antagonist) at any one or more ofthe glucagon, GLP-1, GLP-2, and GIP receptors and comprise an amino acidsequence that shares at least 20% sequence identity (e.g., 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) with atleast one of native glucagon, native oxyntomodulin, native exendin-4,native GLP-1(7-37), native GLP-2, or native GIP over the length of thepeptide (or over the positions which correspond to glucagon, see e.g.FIG. 1).

It is understood that all possible activity subsets of glucagon relatedpeptides are contemplated, e.g. peptides which have biological activity(as agonists or antagonists) at any one or more of the glucagon or GLP-1or GIP receptors, together with all possible subsets of sequenceidentity to each listed native peptide, e.g., comprise an amino acidsequence that shares at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with nativeGLP-1 over the length of native GLP-1. In some embodiments of theinvention, the glucagon related peptide is a peptide having glucagonreceptor agonist activity, GLP-1 receptor agonist activity, GIP receptoragonist activity, glucagon receptor/GLP-1 receptor co-agonist activity,glucagon receptor/GIP receptor co-agonist activity, GLP-1 receptor/GIPreceptor co-agonist activity, glucagon receptor/GLP-1 receptor/GIPreceptor tri-agonist activity, glucagon receptor antagonist activity, orglucagon receptor antagonist/GLP-1 receptor agonist activity. In someembodiments, the peptide retains an alpha-helix conformation in theC-terminal half of the molecule. In some embodiments, the peptideretains positions involved in receptor interaction or signaling, e.g.position 3 of glucagon, or position 7, 10, 12, 13, 15 or 17 ofGLP-1(7-37). Accordingly, the glucagon related peptide can be a peptideof Class 1, Class 2, Class 3, Class 4, and/or Class 5, each of which isfurther described herein.

Q may also be any of the glucagon superfamily peptides that are known inthe art, some of which are disclosed herein by way of nonlimitingexamples. A variety of GLP-1 analogs are known in the art and are aglucagon-related peptide according to the current invention, see, e.g.,WO 2008023050, WO 2007030519, WO 2005058954, WO 2003011892, WO2007046834, WO 2006134340, WO 2006124529, WO 2004022004, WO 2003018516,WO 2007124461 each incorporated herein by reference in its entirety foreach of its sequence or formula disclosures of GLP-1 analogs orderivatives. In any of the embodiments, Q can be a glucagon relatedpeptide disclosed in WO 2007/056362, WO 2008/086086, WO 2009/155527, WO2008/101017, WO 2009/155258, WO 2009/058662, WO 2009/058734, WO2009/099763, WO 2010/011439, PCT Patent Application No. US09/68745, andU.S. Patent Application No. 61/187,578 each incorporated herein byreference in its entirety. In certain embodiments, Q is a Class 1, Class2, Class 3, Class 4, or Class 5 glucagon related peptide as detailedherein. In any of the embodiments described herein, Q is any of SEQ IDNOs: 1-760, 801-919, 1001-1275, 1301-1371, 1401-1518, 1601-1650.

Activity of the Glucagon Superfamily Peptide (Q)

Activity at the Glucagon Receptor

In some embodiments, Q exhibits an EC₅₀ for glucagon receptor activation(or an IC₅₀ for glucagon receptor antagonism) of about 10 mM or less, orabout 1 mM (1000 μM) or less (e.g., about 750 μM or less, about 500 μMor less, about 250 μM or less, about 100 μM or less, about 75 μM orless, about 50 μM or less, about 25 μM or less, about 10 μM or less,about 7.5 μM or less, about 6 μM or less, about 5 μM or less, about 4 μMor less, about 3 μM or less, about 2 μM or less or about 1 μM or less).In some embodiments, Q exhibits an EC₅₀ or IC₅₀ at the glucagon receptorof about 1000 nM or less (e.g., about 750 nM or less, about 500 nM orless, about 250 nM or less, about 100 nM or less, about 75 nM or less,about 50 nM or less, about 25 nM or less, about 10 nM or less, about 7.5nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less,about 3 nM or less, about 2 nM or less or about 1 nM or less). In someembodiments, Q has an EC₅₀ or IC₅₀ at the glucagon receptor which is inthe picomolar range. Accordingly, in some embodiments, Q exhibits anEC₅₀ or IC₅₀ at the glucagon receptor of about 1000 pM or less (e.g.,about 750 pM or less, about 500 pM or less, about 250 pM or less, about100 pM or less, about 75 pM or less, about 50 pM or less, about 25 pM orless, about 10 pM or less, about 7.5 pM or less, about 6 pM or less,about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pMor less or about 1 pM or less).

In some embodiments, Q exhibits an EC₅₀ or IC₅₀ at the glucagon receptorthat is about 0.001 pM or more, about 0.01 pM or more, or about 0.1 pMor more. Glucagon receptor activation (glucagon receptor activity) canbe measured by in vitro assays measuring cAMP induction in HEK293 cellsover-expressing the glucagon receptor, e.g., assaying HEK293 cellsco-transfected with DNA encoding the glucagon receptor and a luciferasegene linked to cAMP responsive element as described in Example 2.

In some embodiments, Q exhibits about 0.001% or more, about 0.01% ormore, about 0.1% or more, about 0.5% or more, about 1% or more, about 5%or more, about 10% or more, about 20% or more, about 30% or more, about40% or more, about 50% or more, about 60% or more, about 75% or more,about 100% or more, about 125% or more, about 150% or more, about 175%or more, about 200% or more, about 250% or more, about 300% or more,about 350% or more, about 400% or more, about 450% or more, or about500% or higher activity at the glucagon receptor relative to nativeglucagon (glucagon potency). In some embodiments, Q exhibits about 5000%or less or about 10,000% or less activity at the glucagon receptorrelative to native glucagon. The activity of Q at a receptor relative toa native ligand of the receptor is calculated as the inverse ratio ofEC₅₀s for Q versus the native ligand.

In some embodiments, Q exhibits substantial activity (potency) at onlythe glucagon receptor and little to no activity at the GLP-1 receptor orthe GIP receptor. In some embodiments, Q is considered as a “pureglucagon receptor agonist” or is not considered as a “glucagon/GLP-1receptor co-agonist” or a “glucagon/GIP receptor co-agonist.” In someembodiments Q exhibits any of the levels of activity or potency at theglucagon receptor described herein but has substantially less activity(potency) at the GLP-1 receptor or the GIP receptor. In someembodiments, Q exhibits an EC₅₀ at the GLP-1 receptor which is 100-foldor greater than the EC₅₀ at the glucagon receptor. In some embodiments,Q exhibits an EC₅₀ at the GIP receptor which is 100-fold or greater thanthe EC₅₀ at the glucagon receptor.

Activity at the GLP-1 Receptor

In some embodiments, Q exhibits an EC₅₀ for GLP-1 receptor activation(or an IC₅₀ for GLP-1 receptor antagonism) of about 10 mM or less, orabout 1 mM (1000 μM) or less (e.g., about 750 μM or less, about 500 μMor less, about 250 μM or less, about 100 μM or less, about 75 μM orless, about 50 μM or less, about 25 μM or less, about 10 μM or less,about 7.5 μM or less, about 6 μM or less, about 5 μM or less, about 4 μMor less, about 3 μM or less, about 2 μM or less or about 1 μM or less).In some embodiments, Q exhibits an EC₅₀ or IC₅₀ for GLP-1 receptoractivation of about 1000 nM or less (e.g., about 750 nM or less, about500 nM or less, about 250 nM or less, about 100 nM or less, about 75 nMor less, about 50 nM or less, about 25 nM or less, about 10 nM or less,about 7.5 nM or less, about 6 nM or less, about 5 nM or less, about 4 nMor less, about 3 nM or less, about 2 nM or less or about 1 nM or less).In some embodiments, Q has an EC₅₀ or IC₅₀ at the GLP-1 receptor whichis in the picomolar range. Accordingly, in some embodiments, Q exhibitsan EC₅₀ or IC₅₀ for GLP-1 receptor activation of about 1000 pM or less(e.g., about 750 pM or less, about 500 pM or less, about 250 pM or less,about 100 pM or less, about 75 pM or less, about 50 pM or less, about 25pM or less, about 10 pM or less, about 7.5 pM or less, about 6 pM orless, about 5 pM or less, about 4 pM or less, about 3 pM or less, about2 pM or less or about 1 pM or less).

In some embodiments, Q exhibits an EC₅₀ or IC₅₀ at the GLP-1 receptorthat is about 0.001 pM or more, about 0.01 pM or more, or about 0.1 pMor more. GLP-1 receptor activation (GLP-1 receptor activity) can bemeasured by in vitro assays measuring cAMP induction in HEK293 cellsover-expressing the GLP-1 receptor, e.g., assaying HEK293 cellsco-transfected with DNA encoding the GLP-1 receptor and a luciferasegene linked to cAMP responsive element as described in Example 2.

In some embodiments, Q exhibits about 0.001% or more, about 0.01% ormore, about 0.1% or more, about 0.5% or more, about 1% or more, about 5%or more, about 10% or more, about 20% or more, about 30% or more, about40% or more, about 50% or more, about 60% or more, about 75% or more,about 100% or more, about 125% or more, about 150% or more, about 175%or more, about 200% or more, about 250% or more, about 300% or more,about 350% or more, about 400% or more, about 450% or more, or about500% or higher activity at the GLP-1 receptor relative to native GLP-1(GLP-1 potency). In some embodiments, Q exhibits about 5000% or less orabout 10,000% or less activity at the GLP-1 receptor relative to nativeGLP-1 (GLP-1 potency).

In some embodiments, Q exhibit substantial activity (potency) at onlythe GLP-1 receptor and little to no activity at the glucagon receptor orthe GIP receptor. In some embodiments, Q is considered as a “pure GLP-1receptor agonist” or is not considered as a “glucagon/GLP-1 receptorco-agonist” or a “GLP-1/GIP co-agonist.” In some embodiments Q exhibitsany of the levels of activity or potency at the GLP-1 receptor describedherein but have substantially less activity (potency) at the glucagonreceptor or the GIP receptor. In some embodiments, Q exhibits an EC₅₀ atthe glucagon receptor which is 100-fold or greater than the EC₅₀ at theGLP-1 receptor. In some embodiments, Q exhibits an EC₅₀ at the GIPreceptor which is 100-fold or greater than the EC₅₀ at the GLP-1receptor.

Activity at the GIP Receptor

In some embodiments, Q exhibits an EC₅₀ for GIP receptor activation (oran IC₅₀ for GIP receptor antagonism) of about 10 mM or less, or about 1mM (1000 μM) or less (e.g., about 750 μM or less, about 500 μM or less,about 250 μM or less, about 100 μM or less, about 75 μM or less, about50 μM or less, about 25 μM or less, about 10 μM or less, about 7.5 μM orless, about 6 μM or less, about 5 μM or less, about 4 μM or less, about3 μM or less, about 2 μM or less or about 1 μM or less). In someembodiments, the EC₅₀ or IC₅₀ of Q at the GIP receptor is less than 1000nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600nM, less than 500 nM, less than 400 nM, less than 300 nM, or less than200 nM. In some embodiments, the EC₅₀ or IC₅₀ of Q at the GIP receptoris about 100 nM or less, e.g., about 75 nM or less, about 50 nM or less,about 25 nM or less, about 10 nM or less, about 8 nM or less, about 6 nMor less, about 5 nM or less, about 4 nM or less, about 3 nM or less,about 2 nM or less, or about 1 nM or less. In some embodiments, the Qexhibits an EC₅₀ or IC₅₀ for GIP receptor activation which is in thepicomolar range. In exemplary embodiments, the EC₅₀ or IC₅₀ of Q at theGIP receptor is less than 1000 pM, less than 900 pM, less than 800 pM,less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM,less than 300 pM, less than 200 pM. In some embodiments, the EC₅₀ orIC₅₀ of Q at the GIP receptor is about 100 pM or less, e.g., about 75 pMor less, about 50 pM or less, about 25 pM or less, about 10 pM or less,about 8 pM or less, about 6 pM or less, about 5 pM or less, about 4 pMor less, about 3 pM or less, about 2 pM or less, or about 1 pM or less.Receptor activation can be measured by in vitro assays measuring cAMPinduction in HEK293 cells over-expressing the GIP receptor, e.g.assaying HEK293 cells co-transfected with DNA encoding the receptor anda luciferase gene linked to cAMP responsive element as described inExample 2.

In some embodiments of the present disclosures, Q exhibits at least orabout 0.1% activity of native GIP at the GIP receptor. In exemplaryembodiments, Q exhibits at least or about 0.2%, at least or about 0.3%,at least or about 0.4%, at least or about 0.5%, at least or about 0.6%,at least or about 0.7%, at least or about 0.8%, at least or about 0.9%,at least or about 1%, at least or about 5%, at least or about 10%, atleast or about 20%, at least or about 30%, at least or about 40%, atleast or about 50%, at least or about 60%, at least or about 70%, atleast or about 75%, at least or about 80%, at least or about 90%, atleast or about 95%, or at least or about 100% of the activity of nativeGIP at the GIP receptor.

In some embodiments of the present disclosures, Q exhibits activity atthe GIP receptor which is greater than that of native GIP. In exemplaryembodiments, Q exhibits at least or about 101%, at least or about 105%,at least or about 110%, at least or about 125%, at least or about 150%,at least or about 175% at least or about 200%, at least or about 300%,at least or about 400%, at least or about 500% or higher % of theactivity of native GIP at the GIP receptor. In some embodiments, Qexhibits no more than 1000%, 10,000%, 100,000%, or 1,000,000% activityat the GIP receptor relative to native GIP. A peptide's activity at theGIP receptor relative to native GIP is calculated as the inverse ratioof EC50s for the GIP agonist peptide vs. native GIP.

In some embodiments, Q exhibits substantial activity (potency) at onlythe GIP receptor and little to no activity at the glucagon receptor orthe GLP-1 receptor. In some embodiments, Q is considered as a “pure GIPreceptor agonist” or is not considered as a “glucagon/GIP receptorco-agonist” or a “GLP-1/GIP co-agonist.” In some embodiments Q exhibitsany of the levels of activity or potency at the GIP receptor describedherein but has substantially less activity (potency) at the glucagonreceptor or the GLP-1 receptor. In some embodiments, Q exhibits an EC₅₀at the glucagon receptor which is 100-fold or greater than the EC₅₀ atthe GIP receptor and an EC₅₀ at the GLP-1 receptor which is 100-fold orgreater than the EC₅₀ at the GIP receptor.

Activity at the GLP-1 Receptor and the Glucagon Receptor

In some embodiments, Q exhibits activity at both the GLP-1 receptor andglucagon receptor (“glucagon/GLP-1 receptor co-agonists”). In someembodiments, the activity (e.g., the EC₅₀ or the relative activity orpotency) of Q at the glucagon receptor is within about 50-fold, about40-fold, about 30-fold, about 20-fold, about 10-fold, or about 5 folddifferent (higher or lower) from its activity (e.g., the EC₅₀ or therelative activity or potency) at the GLP-1 receptor. In someembodiments, the glucagon potency of Q is within about 25-, about 20-,about 15-, about 10-, or about 5-fold different (higher or lower) fromits GLP-1 potency.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the glucagon receptor divided by the relativeactivity or the EC₅₀ or potency of Q at the GLP-1 receptor is less than,or is about, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20,15, 10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the glucagon receptor divided by the EC₅₀ orpotency or relative activity of Q at the GLP-1 receptor is about 1 lessthan 5 (e.g., about 4, about 3, about 2, about 1). In some embodiments,the ratio of the glucagon potency of Q compared to the GLP-1 potency ofQ is less than, or is about, Z, wherein Z is selected from 100, 75, 60,50, 40, 30, 20, 15, 10, and 5. In some embodiments, the ratio of theglucagon potency of Q compared to the GLP-1 potency of Q is less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, Q hasan EC₅₀ at the glucagon receptor which is 2- to 10-fold (e.g., 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greaterthan the EC₅₀ at the GLP-1 receptor.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Q at the GLP-1 receptor divided by the relative activity orpotency or the EC₅₀ of the glucagon analog at the glucagon receptor isless than, or is about, V, wherein V is selected from 100, 75, 60, 50,40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the EC₅₀ orpotency or relative activity of Q at the GLP-1 receptor divided by theEC₅₀ or potency or relative activity of Q at the glucagon receptor isless than 5 (e.g., about 4, about 3, about 2, about 1). In someembodiments, the ratio of the GLP-1 potency of Q compared to theglucagon potency of Q is less than, or is about, W, wherein W isselected from 100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In someembodiments, the ratio of the GLP-1 potency of Q compared to theglucagon potency of Q is less than 5 (e.g., about 4, about 3, about 2,about 1). In some embodiments, Q has an EC₅₀ at the GLP-1 receptor whichis about 2- to about 10-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greater than the EC₅₀ at theglucagon receptor.

In some embodiments, Q exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of native GLP-1 at the GLP-1 receptor (GLP-1 potency) andexhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeglucagon at the glucagon receptor (glucagon potency).

Selectivity of Q for the glucagon receptor versus the GLP-1 receptor canbe described as the relative ratio of glucagon/GLP-1 activity (Q'sactivity at the glucagon receptor relative to native glucagon, dividedby the analog's activity at the GLP-1 receptor relative to nativeGLP-1). For example, a Q that exhibits 60% of the activity of nativeglucagon at the glucagon receptor and 60% of the activity of nativeGLP-1 at the GLP-1 receptor has a 1:1 ratio of glucagon/GLP-1 activity.Exemplary ratios of glucagon/GLP-1 activity include about 1:1, 1.5:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, or about 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.5. As an example, a glucagon/GLP-1activity ratio of 10:1 indicates a 10-fold selectivity for the glucagonreceptor versus the GLP-1 receptor. Similarly, a GLP-1/glucagon activityratio of 10:1 indicates a 10-fold selectivity for the GLP-1 receptorversus the glucagon receptor.

In some embodiments, Q exhibits substantial activity (potency) at theglucagon receptor and GLP-1 receptor and little to no activity at theGIP receptor. In some embodiments Q exhibits any of the levels ofactivity or potency at the glucagon receptor and the GLP-1 receptordescribed herein but has substantially less activity (potency) at theGIP receptor. In some embodiments, Q exhibits an EC₅₀ at the GIPreceptor which is 100-fold or greater than the EC₅₀ at the glucagonreceptor and the EC₅₀ at the GLP-1 receptor.

Activity at the GLP-1 Receptor and the GIP Receptor

In some embodiments, Q exhibits activity at both the GLP-1 receptor andGIP receptor (“GIP/GLP-1 receptor co-agonists”). In some embodiments,the activity (e.g., the EC₅₀ or the relative activity or potency) of Qat the GIP receptor is within about 50-fold, about 40-fold, about30-fold, about 20-fold, about 10-fold, or about 5 fold different (higheror lower) from its activity (e.g., the EC₅₀ or the relative activity orpotency) at the GLP-1 receptor. In some embodiments, the GIP potency ofQ is within about 25-, about 20-, about 15-, about 10-, or about 5-folddifferent (higher or lower) from its GLP-1 potency.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the GIP receptor divided by the relativeactivity or the EC₅₀ or potency of Q at the GLP-1 receptor is less than,or is about, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20,15, 10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the GIP receptor divided by the EC₅₀ orpotency or relative activity of Q at the GLP-1 receptor is about 1 lessthan 5 (e.g., about 4, about 3, about 2, about 1). In some embodiments,the ratio of the GIP potency of Q compared to the GLP-1 potency of Q isless than, or is about, Z, wherein Z is selected from 100, 75, 60, 50,40, 30, 20, 15, 10, and 5. In some embodiments, the ratio of the GIPpotency of Q compared to the GLP-1 potency of Q is less than 5 (e.g.,about 4, about 3, about 2, about 1). In some embodiments, Q has an EC₅₀at the GIP receptor which is 2- to 10-fold (e.g., 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greater thanthe EC₅₀ at the GLP-1 receptor.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Q at the GLP-1 receptor divided by the relative activity orpotency or the EC₅₀ of Q at the GIP receptor is less than, or is about,V, wherein V is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.In some embodiments, the ratio of the EC₅₀ or potency or relativeactivity of Q at the GLP-1 receptor divided by the EC₅₀ or potency orrelative activity of Q at the GIP receptor is less than 5 (e.g., about4, about 3, about 2, about 1). In some embodiments, the ratio of theGLP-1 potency of Q compared to the GIP potency of Q is less than, or isabout, W, wherein W is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, and 5. In some embodiments, the ratio of the GLP-1 potency of Qcompared to the GIP potency of Q is less than 5 (e.g., about 4, about 3,about 2, about 1). In some embodiments, Q has an EC₅₀ at the GLP-1receptor which is about 2- to about 10-fold (e.g., 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greater thanthe EC₅₀ at the GIP receptor.

In some embodiments, Q exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of native GLP-1 at the GLP-1 receptor (GLP-1 potency) andexhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeGIP at the GIP receptor (GIP potency).

Selectivity of Q for the GIP receptor versus the GLP-1 receptor can bedescribed as the relative ratio of GIP/GLP-1 activity (Q's activity atthe GIP receptor relative to native GIP, divided by the analog'sactivity at the GLP-1 receptor relative to native GLP-1). For example, aQ that exhibits 60% of the activity of native GIP at the GIP receptorand 60% of the activity of native GLP-1 at the GLP-1 receptor has a 1:1ratio of GIP/GLP-1 activity. Exemplary ratios of GIP/GLP-1 activityinclude about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or10:1, or about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.5.As an example, a GIP/GLP-1 activity ratio of 10:1 indicates a 10-foldselectivity for the GIP receptor versus the GLP-1 receptor. Similarly, aGLP-1/GIP activity ratio of 10:1 indicates a 10-fold selectivity for theGLP-1 receptor versus the GIP receptor.

Activity at the Glucagon Receptor and the GIP Receptor

In some embodiments, Q exhibits activity at both the glucagon receptorand GIP receptor (“GIP/glucagon receptor co-agonists”). In someembodiments, the activity (e.g., the EC₅₀ or the relative activity orpotency) of Q at the GIP receptor is within about 50-fold, about40-fold, about 30-fold, about 20-fold, about 10-fold, or about 5 folddifferent (higher or lower) from its activity (e.g., the EC₅₀ or therelative activity or potency) at the glucagon receptor. In someembodiments, the GIP potency of Q is within about 25-, about 20-, about15-, about 10-, or about 5-fold different (higher or lower) from itsglucagon potency.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the GIP receptor divided by the relativeactivity or the EC₅₀ or potency of Q at the glucagon receptor is lessthan, or is about, X, wherein X is selected from 100, 75, 60, 50, 40,30, 20, 15, 10, or 5. In some embodiments, the ratio of the EC₅₀ orpotency or relative activity of Q at the GIP receptor divided by theEC₅₀ or potency or relative activity of Q at the glucagon receptor isabout 1 less than 5 (e.g., about 4, about 3, about 2, about 1). In someembodiments, the ratio of the GIP potency of Q compared to the glucagonpotency of Q is less than, or is about, Z, wherein Z is selected from100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In some embodiments, theratio of the GIP potency of Q compared to the glucagon potency of Q isless than 5 (e.g., about 4, about 3, about 2, about 1). In someembodiments, Q has an EC₅₀ at the GIP receptor which is 2- to 10-fold(e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold) greater than the EC₅₀ at the glucagon receptor.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Q at the glucagon receptor divided by the relative activityor potency or the EC₅₀ of Q at the GIP receptor is less than, or isabout, V, wherein V is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the glucagon receptor divided by the EC₅₀ orpotency or relative activity of Q at the GIP receptor is less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, theratio of the glucagon potency of Q compared to the GIP potency of Q isless than, or is about, W, wherein W is selected from 100, 75, 60, 50,40, 30, 20, 15, 10, and 5. In some embodiments, the ratio of theglucagon potency of Q compared to the GIP potency of Q is less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, Q hasan EC₅₀ at the glucagon receptor which is about 2- to about 10-fold(e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold) greater than the EC₅₀ at the GIP receptor.

In some embodiments, Q exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of native glucagon at the glucagon receptor (glucagon potency)and exhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeGIP at the GIP receptor (GIP potency).

Selectivity of Q for the GIP receptor versus the glucagon receptor canbe described as the relative ratio of GIP/glucagon activity (Q'sactivity at the GIP receptor relative to native GIP, divided by theanalog's activity at the glucagon receptor relative to native glucagon).For example, a Q that exhibits 60% of the activity of native GIP at theGIP receptor and 60% of the activity of native glucagon at the glucagonreceptor has a 1:1 ratio of GIP/glucagon activity. Exemplary ratios ofGIP/glucagon activity include about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1 or 10:1, or about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3,1:2, or 1:1.5. As an example, a GIP/glucagon activity ratio of 10:1indicates a 10-fold selectivity for the GIP receptor versus the glucagonreceptor. Similarly, a glucagon/GIP activity ratio of 10:1 indicates a10-fold selectivity for the glucagon receptor versus the GIP receptor.

Activity at the Glucagon Receptor, the GLP-1 Receptor, and the GIPReceptor

In some embodiments, Q exhibits activity at all three of the glucagonreceptor, the GLP-1 receptor, and the GIP receptor (“glucagon/GLP-1/GIPreceptor tri-agonists”). In some embodiments, the activity (e.g., theEC₅₀ or the relative activity or potency) of Q at the glucagon receptoris within about 100-fold, about 75-fold, about 60-fold, 50-fold, about40-fold, about 30-fold, about 20-fold, about 10-fold, or about 5 folddifferent (higher or lower) from its activity (e.g., the EC₅₀ or therelative activity or potency) at both the GLP-1 receptor and the GIPreceptor. In some embodiments, the activity (e.g., the EC₅₀ or therelative activity or potency) of Q at the GLP-1 receptor is within about100-fold, about 75-fold, about 60-fold, 50-fold, about 40-fold, about30-fold, about 20-fold, about 10-fold, or about 5 fold different (higheror lower) from its activity (e.g., the EC₅₀ or the relative activity orpotency) at both the glucagon receptor and the GIP receptor. In someembodiments, the activity (e.g., the EC₅₀ or the relative activity orpotency) of Q at the GIP receptor is within about 100-fold, about75-fold, about 60-fold, 50-fold, about 40-fold, about 30-fold, about20-fold, about 10-fold, or about 5 fold different (higher or lower) fromits activity (e.g., the EC₅₀ or the relative activity or potency) atboth the glucagon receptor and the GLP-1 receptor. This fold differencecan be alternatively expressed as ratios of glucagon/GLP-1, orGLP-1/GIP, or glucagon/GLP-1 as above.

Structure of the Glucagon Superfamily Peptide (Q)

The glucagon superfamily peptide (Q) described herein can comprise anamino acid sequence which is based on the amino acid sequence of nativehuman glucagon (SEQ ID NO: 1601), native human GLP-1 (SEQ ID NOs: 1603or 1604), or native human GIP (SEQ ID NO: 1607).

Based on Native Human Glucagon

In some aspects of the invention, the glucagon superfamily peptide (Q)comprises an amino acid sequence that is based on the amino acidsequence of native human glucagon (SEQ ID NO: 1601). In some aspects, Qcomprises a modified amino acid sequence of SEQ ID NO: 1601 comprising1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in someinstances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.),amino acid modifications. In some embodiments, Q comprises a total of 1,up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9,or up to 10 amino acid modifications relative to the native humanglucagon sequence (SEQ ID NO: 1601). In some embodiments, themodifications are any of those described herein, e.g., acylation,alkylation, pegylation, truncation at C-terminus, substitution of theamino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17,18, 19, 20, 21, 23, 24, 27, 28, and 29.

In some embodiments, Q comprises an amino acid sequence which has atleast 25% sequence identity to the amino acid sequence of native humanglucagon (SEQ ID NO: 1601). In some embodiments, Q comprises an aminoacid sequence which is at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 1601. In someembodiments, the amino acid sequence of Q, which has theabove-referenced % sequence identity is the full-length amino acidsequence of Q. In some embodiments, the amino acid sequence of Q whichhas the above-referenced % sequence identity is only a portion of theamino acid sequence of Q. In some embodiments, Q comprises an amino acidsequence which has about A % or greater sequence identity to a referenceamino acid sequence of at least 5 contiguous amino acids (e.g., at least6, at least 7, at least 8, at least 9, at least 10 amino acids) of SEQID NO: 1601, wherein the reference amino acid sequence begins with theamino acid at position C of SEQ ID NO: 1601 and ends with the amino acidat position D of SEQ ID NO: 1601, wherein A is 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29.Any and all possible combinations of the foregoing parameters areenvisioned, including but not limited to, e.g., wherein A is 90% and Cand D are 1 and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and27, or 16 and 27.

Based on Native Human GLP-1

In some aspects of the invention, the glucagon superfamily peptide (Q)comprises an amino acid sequence that is based on the amino acidsequence of native human GLP-1 (SEQ ID NO: 1603). In some aspects, Qcomprises a modified amino acid sequence of SEQ ID NO: 1603 comprising1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and in someinstances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.),amino acid modifications. In some embodiments, Q comprises a total of 1,up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9,or up to 10 amino acid modifications relative to the native human GLP-1sequence (SEQ ID NO: 1603). In some embodiments, the modifications areany of those described herein, e.g., acylation, alkylation, pegylation,truncation at C-terminus, substitution of the amino acid at one or moreof positions 1, 2, 3, 7, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27,28, and 29.

In some embodiments, Q comprises an amino acid sequence which has atleast 25% sequence identity to the amino acid sequence of native humanGLP-1 (SEQ ID NO: 1603). In some embodiments, Q comprises an amino acidsequence which is at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 1603. In someembodiments, the amino acid sequence of Q, which has theabove-referenced % sequence identity is the full-length amino acidsequence of Q. In some embodiments, the amino acid sequence of Q whichhas the above-referenced % sequence identity is only a portion of theamino acid sequence of Q. In some embodiments, Q comprises an amino acidsequence which has about A % or greater sequence identity to a referenceamino acid sequence of at least 5 contiguous amino acids (e.g., at least6, at least 7, at least 8, at least 9, at least 10 amino acids) of SEQID NO: 1603, wherein the reference amino acid sequence begins with theamino acid at position C of SEQ ID NO: 1603 and ends with the amino acidat position D of SEQ ID NO: 1603, wherein A is 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29.Any and all possible combinations of the foregoing parameters areenvisioned, including but not limited to, e.g., wherein A is 90% and Cand D are 1 and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and27, or 16 and 27.

Based on Native Human GIP

In some embodiments of the present disclosures, Q is an analog of nativehuman GIP, the amino acid sequence of which is provided herein as SEQ IDNO: 1607. Accordingly, in some embodiments, Q comprises an amino acidsequence which is based on the amino acid sequence of SEQ ID NO: 1607but is modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,and in some instances, 16 or more (e.g., 17, 18, 19, 20, 21, 22, 23, 24,25, etc.), amino acid modifications. In some embodiments, Q comprises atotal of 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to8, up to 9, or up to 10 amino acid modifications relative to the nativehuman GIP sequence (SEQ ID NO: 1607). In some embodiments, themodifications are any of those described herein, e.g., acylation,alkylation, pegylation, truncation at C-terminus, substitution of theamino acid at one or more of positions 1, 2, 3, 7, 10, 12, 15, 16, 17,18, 19, 20, 21, 23, 24, 27, 28, and 29. Exemplary GIP receptor agonistsare known in the art. See, for example, Irwin et al., J Pharm and ExpmtTher 314(3): 1187-1194 (2005); Salhanick et al., Bioorg Med Chem Lett15(18): 4114-4117 (2005); Green et al., Diabetes 7(5): 595-604 (2005);O'Harte et al., J Endocrinol 165(3): 639-648 (2000); O'Harte et al.,Diabetologia 45(9): 1281-1291 (2002); Gault et al., Biochem J 367 (Pt3):913-920 (2002); Gault et al., J Endocrin 176: 133-141 (2003); Irwin etal., Diabetes Obes Metab. 11(6): 603-610 (epub 2009).

In some embodiments, Q comprises an amino acid sequence which has atleast 25% sequence identity to the amino acid sequence of native humanGIP (SEQ ID NO: 1607). In some embodiments, Q comprises an amino acidsequence which is at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90% or hasgreater than 90% sequence identity to SEQ ID NO: 1607. In someembodiments, the amino acid sequence of Q which has the above-referenced% sequence identity is the full-length amino acid sequence of Q. In someembodiments, the amino acid sequence of Q which has the above-referenced% sequence identity is only a portion of the amino acid sequence of Q.In some embodiments, Q comprises an amino acid sequence which has aboutA % or greater sequence identity to a reference amino acid sequence ofat least 5 contiguous amino acids (e.g., at least 6, at least 7, atleast 8, at least 9, at least 10 amino acids) of SEQ ID NO: 1607,wherein the reference amino acid sequence begins with the amino acid atposition C of SEQ ID NO: 1607 and ends with the amino acid at position Dof SEQ ID NO: 1607, wherein A is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99; C is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, or 28 and D is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29. Any and allpossible combinations of the foregoing parameters are envisioned,including but not limited to, e.g., wherein A is 90% and C and D are 1and 27, or 6 and 27, or 8 and 27, or 10 and 27, or 12 and 27, or 16 and27.

Modifications

When Q is a glucagon related peptide, Q can comprise the native glucagonamino acid sequence (SEQ ID NO: 1601) with modifications. In exemplaryembodiments, the glucagon related peptide may comprise a total of 1, upto 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, orup to 10 amino acid modifications relative to the native glucagonsequence, e.g. conservative or non-conservative substitutions.Modifications and substitutions described herein are, in certain aspectsmade at specific positions within Q wherein the numbering of theposition corresponds to the numbering of glucagon (SEQ ID NO: 1601). Insome embodiments 1, 2, 3, 4 or 5 non-conservative substitutions arecarried out at any of positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19,20, 21, 24, 27, 28 or 29 and up to 5 further conservative substitutionsare carried out at any of these positions. In some embodiments 1, 2, or3 amino acid modifications are carried out within amino acids atpositions 1-16, and 1, 2 or 3 amino acid modifications are carried outwithin amino acids at positions 17-26. In some embodiments, Q retains atleast 22, 23, 24, 25, 26, 27 or 28 of the naturally occurring aminoacids at the corresponding positions in native glucagon (e.g. have 1-7,1-5 or 1-3 modifications relative to naturally occurring glucagon).

DPP-IV Resistance

In some embodiments where Q is a glucagon related peptide, Q comprises amodification at position 1 or 2 to reduce susceptibility to cleavage bydipeptidyl peptidase IV (DPP-IV). More particularly, in someembodiments, position 1 of Q (e.g., selected from those in FIG. 1) issubstituted with an amino acid selected from the group consisting ofD-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA),N-methyl histidine, alpha-methyl histidine, imidazole acetic acid,desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. More particularly, in some embodiments, position 2 of Qis substituted with an amino acid selected from the group consisting ofD-serine, D-alanine, valine, glycine, N-methyl serine, andaminoisobutyric acid. In some embodiments, position 2 of the glucagonrelated peptide is not D-serine.

Glucagon Modification at Position 3

Glucagon related peptides of Classes 1 to 3 described herein may bemodified at position 3 (according to the amino acid numbering of wildtype glucagon) to maintain or increase activity at the glucagonreceptor.

In some embodiments in which Q is a Class 1, Class 2, or Class 3glucagon related peptide, maintained or enhanced activity at theglucagon receptor may be achieved by modifying the Gln at position 3with a glutamine analog. For example, a Class 1, Class 2, or Class 3glucagon related peptide comprising a glutamine analog at position 3 mayexhibit about 5%, about 10%, about 20%, about 50%, or about 85% orgreater the activity of native glucagon (SEQ ID NO: 1601) at theglucagon receptor. In some embodiments a Class 1, Class 2, or Class 3glucagon related peptide comprising a glutamine analog at position 3 mayexhibit about 20%, about 50%, about 75%, about 100%, about 200% or about500% or greater the activity of a corresponding glucagon peptide havingthe same amino acid sequence as the peptide comprising the glutamineanalog, except for the modified amino acid at position 3 at the glucagonreceptor. In some embodiments, a Class 1, Class 2, or Class 3 glucagonrelated peptide comprising a glutamine analog at position 3 exhibitsenhanced activity at the glucagon receptor, but the enhanced activity isno more than 1000%, 10,000%, 100,000%, or 1,000,000% of the activity ofnative glucagon or of a corresponding glucagon related peptide havingthe same amino acid sequence as the peptide comprising the glutamineanalog, except for the modified amino acid at position 3.

In some embodiments, the glutamine analog is a naturally occurring or anon-naturally occurring amino acid comprising a side chain of StructureI, II or III:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃ alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, O, or S; and Y isNHR⁴, SR³, or OR³. In some embodiments, X is NH or Y is NHR⁴. In someembodiments, R¹ is C₀₋₂ alkyl or C₁ heteroalkyl. In some embodiments, R²is NHR⁴ or C₁ alkyl. In some embodiments, R⁴ is H or C¹ alkyl. Inexemplary embodiments in which Q is a Class 1, Class 2, or Class 3glucagon related peptide, an amino acid comprising a side chain ofStructure I is provided where, R¹ is CH₂—S, X is NH, and R² is CH₃(acetamidomethyl-cysteine, C(Acm)); R¹ is CH₂, X is NH, and R² is CH₃(acetyldiaminobutanoic acid, Dab(Ac)); R¹ is C₀ alkyl, X is NH, R² isNHR⁴, and R⁴ is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R¹ isCH₂—CH₂, X is NH, and R² is CH₃ (acetylornithine, Orn(Ac)). In exemplaryembodiments an amino acid comprising a side chain of Structure II isprovided where, R¹ is CH₂, Y is NHR⁴, and R⁴ is CH₃ (methylglutamine,Q(Me)); In exemplary embodiments an amino acid comprising a side chainof Structure IIII is provided where, R¹ is CH₂ and R⁴ is H(methionine-sulfoxide, M(O)); In specific embodiments, the amino acid atposition 3 is substituted with Dab(Ac).Acylation of Q

In some embodiments, the glucagon related peptide (e.g. a Class 1glucagon related peptide, Class 2 glucagon related peptide, Class 3glucagon related peptide, Class 4 glucagon related peptide, Class 4glucagon related peptides or Class 5 glucagon related peptide), Q ismodified to comprise an acyl group. The acyl group can be covalentlylinked directly to an amino acid of the peptide Q, or indirectly to anamino acid of Q via a spacer, wherein the spacer is positioned betweenthe amino acid of Q and the acyl group. Q may be acylated at the sameamino acid position where a hydrophilic moiety is linked, or at adifferent amino acid position. As described herein, Q can be a glucagonsuperfamily peptide, glucagon related peptide, including a Class 1, 2,3, 4 or 5 glucagon related peptide, or osteocalcin, calcitonin, amylin,or an analog, derivative or conjugate thereof. For example, Q may be oneof Class 1, Class 2, Class 3, Class 4 or Class 5, and may comprise anacyl group which is non-native to a naturally-occurring amino acid.Acylation can be carried out at any position within Q. Where Q is aglucagon related peptide, acylation may occur at any position includingany of positions 1-29, a position within a C-terminal extension, or theC-terminal amino acid, provided that the activity exhibited by thenon-acylated glucagon related peptide is retained upon acylation. Forexample, if the unacylated peptide has glucagon agonist activity, thenthe acylated peptide retains the glucagon agonist activity. Also forexample, if the unacylated peptide has glucagon antagonist activity,then the acylated peptide retains the glucagon antagonist activity. Forinstance, if the unacylated peptide has GLP-1 agonist activity, then theacylated peptide retains GLP-1 agonist activity. Nonlimiting examplesinclude acylation at positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19,20, 21, 24, 27, 28, or 29 (according to the amino acid numbering of wildtype glucagon). With regard to Class 1, Class 2, and Class 3 glucagonrelated peptides, acylation may occur at any of positions 5, 7, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 40,41, 42, or 43 (according to the amino acid numbering of wild typeglucagon). Other nonlimiting examples with respect to glucagon relatedpeptides (e.g., Class 1, 2, 3, 4, or 5) include acylation at position 10(according to the amino acid numbering of the wild type glucagon) andpegylation at one or more positions in the C-terminal portion of theglucagon peptide, e.g., position 24, 28 or 29 (according to the aminoacid numbering of the wild type glucagon), within a C-terminalextension, or at the C-terminus (e.g., through adding a C-terminal Cys).

In a specific aspect of the invention, peptide Q (e.g., a glucagonsuperfamily peptide, a glucagon related peptide, a Class 1, 2, 3, 4 or 5glucagon related peptide, or osteocalcin, calcitonin, amylin, or ananalog, derivative or conjugate thereof) is modified to comprise an acylgroup by direct acylation of an amine, hydroxyl, or thiol of a sidechain of an amino acid of Q. In some embodiments, Q is directly acylatedthrough the side chain amine, hydroxyl, or thiol of an amino acid. Insome embodiments, where Q is a glucagon related peptide, acylation is atposition 10, 20, 24, or 29 (according to the amino acid numbering of thewild type glucagon). In this regard, the acylated glucagon relatedpeptide can comprise the amino acid sequence of SEQ ID NO: 1601, or amodified amino acid sequence thereof comprising one or more of the aminoacid modifications described herein, with at least one of the aminoacids at positions 10, 20, 24, and 29 (according to the amino acidnumbering of the wild type glucagon) modified to any amino acidcomprising a side chain amine, hydroxyl, or thiol. In some specificembodiments of the invention, where Q is a glucagon related peptide, thedirect acylation of the Q occurs through the side chain amine, hydroxyl,or thiol of the amino acid at position 10 (according to the amino acidnumbering of the wild type glucagon).

In some embodiments, the amino acid of peptide Q (e.g., a glucagonsuperfamily peptide, a glucagon related peptide, a Class 1, 2, 3, 4 or 5glucagon related peptide, or osteocalcin, calcitonin, amylin, or ananalog, derivative or conjugate thereof) comprising a side chain amineis an amino acid of Formula I:

In some exemplary embodiments, the amino acid of Formula I, is the aminoacid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid of peptide Q comprising a sidechain hydroxyl is an amino acid of Formula II:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Ser).

In yet other embodiments, the amino acid of peptide Q comprising a sidechain thiol is an amino acid of Formula III:

In some exemplary embodiments, the amino acid of Formula III is theamino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid of peptide Q comprising a sidechain amine, hydroxyl, or thiol is a disubstituted amino acid comprisingthe same structure of Formula I, Formula II, or Formula III, except thatthe hydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In some embodiments of the invention, the acylated peptide Q (e.g., aglucagon superfamily peptide, a glucagon related peptide, a Class 1, 2,3, 4 or 5 glucagon related peptide, or osteocalcin, calcitonin, amylin,or an analog, derivative or conjugate thereof) comprises a spacerbetween the peptide and the acyl group. In some embodiments, Q iscovalently bound to the spacer, which is covalently bound to the acylgroup. In some exemplary embodiments, Q is modified to comprise an acylgroup by acylation of an amine, hydroxyl, or thiol of a spacer, whichspacer (where Q is a glucagon related peptide, e.g., Class 1, 2, 3, 4 or5) is attached to a side chain of an amino acid at position 10, 20, 24,or 29 (according to the amino acid numbering of the wild type glucagon),or at the C-terminal amino acid of the glucagon related peptide. Theamino acid of peptide Q to which the spacer is attached can be any aminoacid comprising a moiety which permits linkage to the spacer. Forexample, an amino acid comprising a side chain —NH₂, —OH, or —COOH(e.g., Lys, Orn, Ser, Asp, or Glu) is suitable. An amino acid of peptideQ (e.g., a singly or doubly α-substituted amino acid) comprising a sidechain —NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) is alsosuitable. In some embodiments where Q is a glucagon related peptide(e.g., Class 1, 2, 3, 4 or 5), the acylated glucagon related peptide cancomprise the amino acid sequence of SEQ ID NO: 1601, or a modified aminoacid sequence thereof comprising one or more of the amino acidmodifications described herein, with at least one of the amino acids atpositions 10, 20, 24, and 29 (according to the amino acid numbering ofthe wild type glucagon) modified to any amino acid comprising a sidechain amine, hydroxyl, or carboxylate.

In some embodiments, the spacer between the peptide Q and the acyl groupis an amino acid comprising a side chain amine, hydroxyl, or thiol, or adipeptide or tripeptide comprising an amino acid comprising a side chainamine, hydroxyl, or thiol. In some embodiments, the amino acid spacer isnot γ-Glu. In some embodiments, the dipeptide spacer is not γ-Glu-γ-Glu.

When acylation occurs through an amine group of the amino acid of thespacer, the acylation can occur through the alpha amine of the aminoacid or a side chain amine. In the instance in which the alpha amine isacylated, the spacer amino acid can be any amino acid. For example, thespacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val,Leu, Ile, Trp, Met, Phe, Tyr. In some embodiments, the spacer amino acidcan be, for example, a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu,Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid,7-aminoheptanoic acid, 8-aminooctanoic acid. Alternatively, the spaceramino acid can be an acidic residue, e.g., Asp and Glu. In the instancein which the side chain amine of the spacer amino acid is acylated, thespacer amino acid is an amino acid comprising a side chain amine, e.g.,an amino acid of Formula I (e.g., Lys or Orn). In this instance, it ispossible for both the alpha amine and the side chain amine of the spaceramino acid to be acylated, such that the peptide is diacylated.Embodiments of the invention include such diacylated molecules.

When acylation occurs through a hydroxyl group of the amino acid of thespacer, the amino acid or one of the amino acids of the dipeptide ortripeptide can be an amino acid of Formula II. In a specific exemplaryembodiment, the amino acid is Ser.

When acylation occurs through a thiol group of the amino acid of thespacer, the amino acid or one of the amino acids of the dipeptide ortripeptide can be an amino acid of Formula III. In a specific exemplaryembodiment, the amino acid is Cys.

In some embodiments, the spacer comprises a hydrophilic bifunctionalspacer. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments the spacer between peptide Q and the acyl groupcomprises a hydrophilic bifunctional spacer. In certain embodiments, thehydrophilic bifunctional spacer comprises two or more reactive groups,e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or anycombinations thereof. In certain embodiments, the hydrophilicbifunctional spacer comprises a hydroxyl group and a carboxylate. Inother embodiments, the hydrophilic bifunctional spacer comprises anamine group and a carboxylate. In other embodiments, the hydrophilicbifunctional spacer comprises a thiol group and a carboxylate.

In some embodiments, the spacer between peptide Q and the acyl group isa hydrophobic bifunctional spacer. Hydrophobic bifunctional spacers areknown in the art. See, e.g., Bioconjugate Techniques, G. T. Hermanson(Academic Press, San Diego, Calif., 1996), which is incorporated byreference in its entirety. In certain embodiments, the hydrophobicbifunctional spacer comprises two or more reactive groups, e.g., anamine, a hydroxyl, a thiol, and a carboxyl group or any combinationsthereof. In certain embodiments, the hydrophobic bifunctional spacercomprises a hydroxyl group and a carboxylate. In other embodiments, thehydrophobic bifunctional spacer comprises an amine group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises a thiol group and a carboxylate. Suitable hydrophobicbifunctional spacers comprising a carboxylate and a hydroxyl group or athiol group are known in the art and include, for example,8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1 to 7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments, wherein Q is a Class 1, Class 2, or Class 3 glucagonrelated peptide, is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8,9, or 10 atoms) in length. In more specific embodiments in which Q is aClass 1, Class 2, or Class 3 glucagon related peptide, the spacer isabout 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl groupis a C12 to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fattyacyl group, such that the total length of the spacer and acyl group is14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, or 28 atoms. In some embodiments, in which Q is a Class 1,Class 2, or Class 3 glucagon related peptide the length of the spacerand acyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain embodiments in which Q is a Class 1, Class 2,or Class 3 glucagon related peptide, the bifunctional spacer can be asynthetic or naturally occurring amino acid (including, but not limitedto, any of those described herein) comprising an amino acid backbonethat is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid,5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoic acid).Alternatively, the spacer attached to the Class 1, Class 2, or Class 3glucagon related peptide can be a dipeptide or tripeptide spacer havinga peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. Each amino acid of the dipeptide or tripeptide spacer attachedto the Class 1, Class 2, or Class 3 glucagon related peptide can be thesame as or different from the other amino acid(s) of the dipeptide ortripeptide and can be independently selected from the group consistingof: naturally-occurring and/or non-naturally occurring amino acids,including, for example, any of the D or L isomers of thenaturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or Lisomers of the non-naturally occurring amino acids selected from thegroup consisting of: β-alanine β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), a-aminobutyric acid (γ-Abu), aminohexanoic acid(ε-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(02)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO2)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), 1-amino-1-cyclohexane carboxylicacid (Acx), aminovaleric acid, beta-cyclopropyl-alanine (Cpa),propargylglycine (Prg), allylglycine (Alg),2-amino-2-cyclohexyl-propanoic acid (2-Cha), tertbutylglycine (Tbg),vinylglycine (Vg), 1-amino-1-cyclopropane carboxylic acid (Acp),1-amino-1-cyclopentane carboxylic acid (Acpe), alkylated3-mercaptopropionic acid, 1-amino-1-cyclobutane carboxylic acid (Acb).

In some embodiments in which Q is a Class 1, Class 2, or Class 3glucagon related peptide, the spacer comprises an overall negativecharge, e.g., comprises one or two negatively charged amino acids. Insome embodiments in which Q is a Class 1, Class 2, or Class 3 glucagonrelated peptide, the dipeptide is not any of the dipeptides of generalstructure A-B, wherein A is selected from the group consisting of Gly,Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe, and Pro, wherein B isselected from the group consisting of Lys, His, Trp. In some embodimentsin which Q is a Class 1, Class 2, or Class 3 glucagon related peptide,the dipeptide spacer is selected from the group consisting of: Ala-Ala,β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyric acid-γ-aminobutyric acid,and γ-Glu-γ-Glu.

The peptide Q can be modified to comprise an acyl group by acylation ofa long chain alkane. In specific aspects, the long chain alkanecomprises an amine, hydroxyl, or thiol group (e.g. octadecylamine,tetradecanol, and hexadecanethiol) which reacts with a carboxyl group,or activated form thereof, of the peptide Q. The carboxyl group, oractivated form thereof, of Q can be part of a side chain of an aminoacid (e.g., glutamic acid, aspartic acid) of Q or can be part of thepeptide backbone.

In certain embodiments, the peptide Q is modified to comprise an acylgroup by acylation of the long chain alkane by a spacer which isattached to Q. In specific aspects, the long chain alkane comprises anamine, hydroxyl, or thiol group which reacts with a carboxyl group, oractivated form thereof, of the spacer. Suitable spacers comprising acarboxyl group, or activated form thereof, are described herein andinclude, for example, bifunctional spacers, e.g., amino acids,dipeptides, tripeptides, hydrophilic bifunctional spacers andhydrophobic bifunctional spacers.

As used herein, the term “activated form of a carboxyl group” refers toa carboxyl group with the general formula R(C═O)X, wherein X is aleaving group and R is Q or the spacer. For example, activated forms ofa carboxyl groups may include, but are not limited to, acyl chlorides,anhydrides, and esters. In some embodiments, the activated carboxylgroup is an ester with a N-hydroxysuccinimide (NHS) leaving group.

With regard to these aspects of the invention, in which a long chainalkane is acylated by the peptide Q or the spacer, the long chain alkanemay be of any size and can comprise any length of carbon chain. The longchain alkane can be linear or branched. In certain aspects, the longchain alkane is a C4 to C30 alkane. For example, the long chain alkanecan be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane,C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24 alkane,C26 alkane, C28 alkane, or a C30 alkane. In some embodiments, the longchain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane, C16alkane, or a C18 alkane.

In some embodiments, an amine, hydroxyl, or thiol group of Q is acylatedwith a cholesterol acid. In a specific embodiment, the peptide is linkedto the cholesterol acid through an alkylated des-amino Cys spacer, i.e.,an alkylated 3-mercaptopropionic acid spacer.

Suitable methods of peptide acylation via amines, hydroxyls, and thiolsare known in the art. See, for example, Miller, Biochem Biophys ResCommun 218: 377-382 (1996); Shimohigashi and Stammer, Int J Pept ProteinRes 19: 54-62 (1982); and Previero et al., Biochim Biophys Acta 263:7-13 (1972) (for methods of acylating through a hydroxyl); and San andSilvius, J Pept Res 66: 169-180 (2005) (for methods of acylating througha thiol); Bioconjugate Chem. “Chemical Modifications of Proteins:History and Applications” pages 1, 2-12 (1990); Hashimoto et al.,Pharmacuetical Res. “Synthesis of Palmitoyl Derivatives of Insulin andtheir Biological Activity” Vol. 6, No: 2 pp. 171-176 (1989).

The acyl group of the acylated peptide Q can be of any size, e.g., anylength carbon chain, and can be linear or branched. In some specificembodiments of the invention, the acyl group is a C4 to C30 fatty acid.For example, the acyl group can be any of a C4 fatty acid, C6 fattyacid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fattyacid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid. In someembodiments, the acyl group is a C8 to C20 fatty acid, e.g., a C14 fattyacid or a C16 fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

The acylated peptides Q described herein can be further modified tocomprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In someembodiments related to Class 1, 2, 3, 4 or 5 glucagon related peptides,the acylated glucagon related peptide can comprise SEQ ID NO: 1601,including any of the modifications described herein, in which at leastone of the amino acids at position 10, 20, 24, and 29 (according to theamino acid numbering of the wild type glucagon) comprise an acyl groupand at least one of the amino acids at position 16, 17, 21, 24, or 29(according to the amino acid numbering of the wild type glucagon), aposition within a C-terminal extension, or the C-terminal amino acid aremodified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the side chain ofthe amino acid is covalently bonded to a hydrophilic moiety (e.g., PEG).In some embodiments related to Class 1, 2, 3, 4 or 5 glucagon relatedpeptides, the acyl group is attached to position 10 (according to theamino acid numbering of the wild type glucagon), optionally via a spacercomprising Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the hydrophilicmoiety is incorporated at a Cys residue at position 24.

Alternatively, the acylated peptide (Q) can comprise a spacer, whereinthe spacer is both acylated and modified to comprise the hydrophilicmoiety. Nonlimiting examples of suitable spacers include a spacercomprising one or more amino acids selected from the group consisting ofCys, Lys, Orn, homo-Cys, and Ac-Phe.

Alkylation of Q

In some embodiments, Q is modified to comprise an alkyl group. The alkylgroup can be covalently linked directly to an amino acid of the peptideQ, or indirectly to an amino acid of Q via a spacer, wherein the spaceris positioned between the amino acid of Q and the alkyl group. The alkylgroup can be attached to Q via an ether, thioether, or amino linkage,for example. Q may be alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position. Asdescribed herein, Q can be a glucagon superfamily peptide, glucagonrelated peptide, including a Class 1, 2, 3, 4 or 5 glucagon relatedpeptide, or osteocalcin, calcitonin, amylin, or an analog, derivative orconjugate thereof. For example, Q may be a Class 1, Class 2, or Class 3glucagon related peptide, and may comprise an alkyl group which isnon-native to a naturally-occurring amino acid.

Alkylation can be carried out at any position within Q. Where Q is aglucagon related peptide, alkylation may occur at any position includingany of positions 1-29, a position within a C-terminal extension, or theC-terminal amino acid, provided that an agonist activity of theunalkyated peptide with respect to glucagon, GLP-1, GIP or otherglucagon-related peptide receptor is retained upon alkylation. In someembodiments, if the unalkylated peptide has glucagon agonist activity,then the alkylated peptide retains glucagon agonist activity isretained. In some embodiments, if the unalkylated peptide has GLP-1agonist activity, then the alkylated peptide retains GLP-1 agonistactivity. Nonlimiting examples include alkylation at positions 5, 7, 10,11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, or 29 (according tothe amino acid numbering of wild type glucagon). With regard to Class 1,Class 2, and Class 3 glucagon related peptides, alkylation can occur atpositions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28,29, 30, 37, 38, 39, 40, 41, 42, or 43 (according to the amino acidnumbering of wild type glucagon). Other nonlimiting examples withrespect to glucagon related peptides (e.g. Class, 1, 2, 3, 4 or 5)include alkylation at position 10 (according to the amino acid numberingof wild type glucagon) and pegylation at one or more positions in theC-terminal portion of the glucagon related peptide, e.g., position 24,28 or 29 (according to the amino acid numbering of wild type glucagon),within a C-terminal extension, or at the C-terminus (e.g., throughadding a C-terminal Cys).

In a specific aspect of the invention, peptide Q (e.g. a glucagonsuperfamily peptide, a glucagon related peptide, a Class 1, 2, 3, 4 or 5glucagon related peptide, or osteocalcin, calcitonin, amylin, or ananalog, derivative or conjugate thereof) is modified to comprise analkyl group by direct alkylation of an amine, hydroxyl, or thiol of aside chain of an amino acid of Q. In some embodiments, Q is directlyalkylated through the side chain amine, hydroxyl, or thiol of an aminoacid. In some embodiments, where Q is a glucagon related peptide,alkylation is at position 10, 20, 24, or 29 (according to the amino acidnumbering of wild type glucagon). In this regard, the alkylated glucagonrelated peptide can comprise the amino acid sequence of SEQ ID NO: 1601,or a modified amino acid sequence thereof comprising one or more of theamino acid modifications described herein, with at least one of theamino acids at positions 10, 20, 24, and 29 (according to the amino acidnumbering of wild type glucagon) modified to any amino acid comprising aside chain amine, hydroxyl, or thiol. In some specific embodiments ofthe invention, where Q is a glucagon related peptide, the directalkylation of Q occurs through the side chain amine, hydroxyl, or thiolof the amino acid at position 10 (according to the amino acid numberingof wild type glucagon).

In some embodiments, the amino acid of peptide Q (e.g. a glucagonsuperfamily peptide, a glucagon related peptide, a Class 1, 2, 3, 4 or 5glucagon related peptide, or osteocalcin, calcitonin, amylin, or ananalog, derivative or conjugate thereof) comprising a side chain amineis an amino acid of Formula I. In some exemplary embodiments, the aminoacid of Formula I, is the amino acid wherein n is 4 (Lys) or n is 3(Orn).

In other embodiments, the amino acid of peptide Q comprising a sidechain hydroxyl is an amino acid of Formula II. In some exemplaryembodiments, the amino acid of Formula II is the amino acid wherein n is1 (Ser).

In yet other embodiments, the amino acid of peptide Q comprising a sidechain thiol is an amino acid of Formula III. In some exemplaryembodiments, the amino acid of Formula II is the amino acid wherein n is1 (Cys).

In yet other embodiments, the amino acid of peptide Q comprising a sidechain amine, hydroxyl, or thiol is a disubstituted amino acid comprisingthe same structure of Formula I, Formula II, or Formula III, except thatthe hydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In some embodiments of the invention, the alkylated peptide Q (e.g. aglucagon superfamily peptide, a glucagon related peptide, a Class 1, 2,3, 4 or 5 glucagon related peptide, or osteocalcin, calcitonin, amylin,or an analog, derivative or conjugate thereof) comprises a spacerbetween the peptide and the alkyl group. In some embodiments, the Q iscovalently bound to the spacer, which is covalently bound to the alkylgroup. In some exemplary embodiments, peptide Q is modified to comprisean alkyl group by alkylation of an amine, hydroxyl, or thiol of aspacer, which spacer (where Q is a glucagon related peptide, e.g., Class1, 2, 3, 4 or 5) is attached to a side chain of an amino acid atposition 10, 20, 24, or 29 (according to the amino acid numbering ofwild type glucagon) of Q. The amino acid of peptide Q to which thespacer is attached can be any amino acid comprising a moiety whichpermits linkage to the spacer. The amino acid of peptide Q to which thespacer is attached can be any amino acid (e.g., a singly α-substitutedamino acid or an α,α-disubstituted amino acid) comprising a moiety whichpermits linkage to the spacer. An amino acid of peptide Q comprising aside chain —NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In some embodiments where Q is a glucagon related peptide(e.g., Class 1, 2, 3, 4 or 5), the alkylated Q can comprise the aminoacid sequence of SEQ ID NO: 1601, or a modified amino acid sequencethereof comprising one or more of the amino acid modifications describedherein, with at least one of the amino acids at positions 10, 20, 24,and 29 (according to the amino acid numbering of wild type glucagon)modified to any amino acid comprising a side chain amine, hydroxyl, orcarboxylate.

In some embodiments, the spacer between the peptide Q and the alkylgroup is an amino acid comprising a side chain amine, hydroxyl, or thiolor a dipeptide or tripeptide comprising an amino acid comprising a sidechain amine, hydroxyl, or thiol. In some embodiments, the amino acidspacer is not γ-Glu. In some embodiments, the dipeptide spacer is notγ-Glu-γ-Glu.

When alkylation occurs through an amine group of the amino acid of thespacer the alkylation can occur through the alpha amine of the aminoacid or a side chain amine. In the instance in which the alpha amine isalkylated, the spacer amino acid can be any amino acid. For example, thespacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val,Leu, Ile, Trp, Met, Phe, Tyr. Alternatively, the spacer amino acid canbe an acidic residue, e.g., Asp and Glu. In exemplary embodiments, thespacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val,Leu, Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovalericacid, 7-aminoheptanoic acid, 8-aminooctanoic acid. Alternatively, thespacer amino acid can be an acidic residue, e.g., Asp and Glu, providedthat the alkylation occurs on the alpha amine of the acidic residue. Inthe instance in which the side chain amine of the spacer amino acid isalkylated, the spacer amino acid is an amino acid comprising a sidechain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn). Inthis instance, it is possible for both the alpha amine and the sidechain amine of the spacer amino acid to be alkylated, such that thepeptide is dialkylated. Embodiments of the invention include suchdialkylated molecules.

When alkylation occurs through a hydroxyl group of the amino acid of thespacer, the amino acid or one of the amino acids of the spacer can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When alkylation occurs through a thiol group of the amino acid of thespacer, the amino acid or one of the amino acids of the spacer can be anamino acid of Formula III. In a specific exemplary embodiment, the aminoacid is Cys.

In some embodiments, the spacer comprises a hydrophilic bifunctionalspacer. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer between peptide Q and the alkyl group isa hydrophilic bifunctional spacer. In certain embodiments, thehydrophilic bifunctional spacer comprises two or more reactive groups,e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or anycombinations thereof. In certain embodiments, the hydrophilicbifunctional spacer comprises a hydroxyl group and a carboxylate. Inother embodiments, the hydrophilic bifunctional spacer comprises anamine group and a carboxylate. In other embodiments, the hydrophilicbifunctional spacer comprises a thiol group and a carboxylate.

In some embodiments, the spacer between peptide Q and the alkyl group isa hydrophobic bifunctional spacer. In certain embodiments, thehydrophobic bifunctional spacer comprises two or more reactive groups,e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or anycombinations thereof. In certain embodiments, the hydrophobicbifunctional spacer comprises a hydroxyl group and a carboxylate. Inother embodiments, the hydropholic bifunctional spacer comprises anamine group and a carboxylate. In other embodiments, the hydropholicbifunctional spacer comprises a thiol group and a carboxylate. Suitablehydrophobic bifunctional spacers comprising a carboxylate and a hydroxylgroup or a thiol group are known in the art and include, for example,8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments in which Q is a Class 1, Class 2, or Class 3 glucagonrelated peptide is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8,9, or 10 atoms)) in length. In more specific embodiments, the spacerattached to the Class 1, Class 2, or Class 3 glucagon related peptide isabout 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is aC12 to C18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, suchthat the total length of the spacer and alkyl group is 14 to 28 atoms,e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or28 atoms. In some embodiments in which Q is a Class 1, Class 2, or Class3 glucagon related peptide, the length of the spacer and alkyl is 17 to28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments in which Q is a Class1, Class 2, or Class 3 glucagon related peptide, the bifunctional spacercan be a synthetic or non-naturally occurring amino acid comprising anamino acid backbone that is 3 to 10 atoms in length (e.g., 6-aminohexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and8-aminooctanoic acid). Alternatively, the spacer attached to the Class1, Class 2, or Class 3 glucagon related peptide can be a dipeptide ortripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g.,6 to 10 atoms) in length. The dipeptide or tripeptide spacer attached tothe Class 1, Class 2, or Class 3 glucagon related peptide can becomposed of naturally-occurring and/or non-naturally occurring aminoacids, including, for example, any of the amino acids taught herein. Insome embodiments in which Q is a Class 1, Class 2, or Class 3 glucagonrelated peptide, the spacer comprises an overall negative charge, e.g.,comprises one or two negatively charged amino acids. In some embodimentsin which Q is a Class 1, Class 2, or Class 3 glucagon related peptide,the dipeptide spacer is selected from the group consisting of: Ala-Ala,β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyric acid-γ-aminobutyric acid,and γGlu-γ-Glu. In some embodiments, the dipeptide spacer is notγ-Glu-γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiolsare known in the art. For example, a Williamson ether synthesis can beused to form an ether linkage between the glucagon related peptide andthe alkyl group. Also, a nucleophilic substitution reaction of thepeptide with an alkyl halide can result in any of an ether, thioether,or amino linkage.

The alkyl group of the alkylated peptide Q can be of any size, e.g., anylength carbon chain, and can be linear or branched. In some embodimentsof the invention, the alkyl group is a C4 to C30 alkyl. For example, thealkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl,C26 alkyl, C28 alkyl, or a C30 alkyl. In some embodiments, the alkylgroup is a C8 to C20 alkyl, e.g., a C14 alkyl or a C16 alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

In some embodiments of the invention, peptide Q is modified to comprisean alkyl group by reacting a nucleophilic, long chain alkane with Q,wherein Q comprises a leaving group suitable for nucleophilicsubstitution. In specific aspects, the nucleophilic group of the longchain alkane comprises an amine, hydroxyl, or thiol group (e.g.octadecylamine, tetradecanol, and hexadecanethiol). The leaving group ofQ can be part of a side chain of an amino acid or can be part of thepeptide backbone. Suitable leaving groups include, for example,N-hydroxysuccinimide, halogens, and sulfonate esters.

In certain embodiments, peptide Q is modified to comprise an alkyl groupby reacting the nucleophilic, long chain alkane with a spacer, which isattached to Q, wherein the spacer comprises the leaving group. Inspecific aspects, the long chain alkane comprises an amine, hydroxyl, orthiol group. In certain embodiments, the spacer comprising the leavinggroup can be any spacer discussed herein, e.g., amino acids, dipeptides,tripeptides, hydrophilic bifunctional spacers and hydrophobicbifunctional spacers further comprising a suitable leaving group.

With regard to these aspects of the invention in which a long chainalkane is alkylated by peptide Q or the spacer, the long chain alkanemay be of any size and can comprise any length of carbon chain. The longchain alkane can be linear or branched. In certain aspects, the longchain alkane is a C4 to C30 alkane. For example, the long chain alkanecan be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12 alkane,C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24 alkane,C26 alkane, C28 alkane, or a C30 alkane. In some embodiments in whichthe glucagon related peptide is a Class 1, Class 2, or Class 3 glucagonrelated peptide, the long chain alkane comprises a C8 to C20 alkane,e.g., a C14 alkane, C16 alkane, or a C18 alkane.

Also, in some embodiments alkylation can occur between Q and acholesterol moiety. For example, the hydroxyl group of cholesterol candisplace a leaving group on the long chain alkane to form acholesterol-glucagon peptide product.

The alkylated peptides (Q) described herein can be further modified tocomprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In someembodiments related to Class 1, 2, 3, 4 or 5 glucagon related peptidesthe alkylated Q can comprise SEQ ID NO: 1601, or a modified amino acidsequence thereof comprising one or more of the amino acid modificationsdescribed herein, in which at least one of the amino acids at position10, 20, 24, and 29 (according to the amino acid numbering of wild typeglucagon) comprise an alkyl group and at least one of the amino acids atposition 16, 17, 21, 24, and 29, a position within a C-terminalextension or the C-terminal amino acid are modified to a Cys, Lys, Orn,homo-Cys, or Ac-Phe, and the side chain of the amino acid is covalentlybonded to a hydrophilic moiety (e.g., PEG). In some embodiments relatedto Class 1, 2, 3, 4 or 5 glucagon related peptides, the alkyl group isattached to position 10 (according to the amino acid numbering of wildtype glucagon), optionally via a spacer comprising Cys, Lys, Orn,homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cysresidue at position 24.

Alternatively, the alkylated peptide Q can comprise a spacer, whereinthe spacer is both alkylated and modified to comprise the hydrophilicmoiety. Nonlimiting examples of suitable spacers include a spacercomprising one or more amino acids selected from the group consisting ofCys, Lys, Orn, homo-Cys, and Ac-Phe.

Stabilization of the Alpha-Helix Structure

In some embodiments, an intramolecular bridge is formed between twoamino acid side chains to stabilize the three dimensional structure ofthe carboxy terminal portion (e.g., amino acids 12-29 (according to theamino acid numbering of wild type glucagon)) of Class 1, 2, 3, 4, or 5glucagon related peptide Q. The two amino acid side chains can be linkedto one another through hydrogen-bonding, ionic interactions, such as theformation of salt bridges, or by covalent bonds.

In some embodiments, the intramolecular bridge is formed between twoamino acids that are 3 amino acids apart, e.g., amino acids at positionsi and i+4, wherein i is any integer between 12 and 25 (e.g., 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) according to the aminoacid numbering of wild type glucagon. More particularly, the side chainsof the amino acid pairs 12 and 16, 16 and 20, 20 and 24 or 24 and 28(amino acid pairs in which i=12, 16, 20, or 24) according to the aminoacid numbering of wild type glucagon are linked to one another and thusstabilize the glucagon alpha helix. Alternatively, i can be 17.

In some specific embodiments, wherein the amino acids at positions i andi+4 are joined by an intramolecular bridge, the size of the linker isabout 8 atoms, or about 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are two amino acids apart, e g, amino acids atpositions j and j+3, wherein j is any integer between 12 and 26 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26)according to the amino acid numbering of wild type glucagon. In somespecific embodiments, j is 17.

In some specific embodiments, wherein amino acids at positions j and j+3are joined by an intramolecular bridge, the size of the linker is about6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are 6 amino acids apart, e.g., amino acids atpositions k and k+7, wherein k is any integer between 12 and 22 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22) according to the aminoacid numbering of wild type glucagon. In some specific embodiments, k is12, 13, or 17. In an exemplary embodiment, k is 17.

Examples of amino acid pairings that are capable of covalently bondingto form a six-atom linking bridge include Orn and Asp, Glu and an aminoacid of Formula I, wherein n is 2, and homoglutamic acid and an aminoacid of Formula I, wherein n is 1, wherein Formula I is:

Examples of amino acid pairing that are capable of covalently bonding toform a seven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modified derivatives that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect. Even without covalentlinkage, the amino acid pairings described above or similar pairingsthat one of ordinary skill in the art can envision may also provideadded stability to the alpha-helix through non-covalent bonds, forexample, through formation of salt bridges or hydrogen-bondinginteractions.

The size of a lactam ring can vary depending on the length of the aminoacid side chains, and in some embodiments the lactam is formed bylinking the side chains of a lysine amino acid to a glutamic acid sidechain. Further exemplary embodiments (according to the amino acidnumbering of wild type glucagon) include the following pairings,optionally with a lactam bridge: Glu at position 12 with Lys at position16; native Lys at position 12 with Glu at position 16; Glu at position16 with Lys at position 20; Lys at position 16 with Glu at position 20;Glu at position 20 with Lys at position 24; Lys at position 20 with Gluat position 24; Glu at position 24 with Lys at position 28; Lys atposition 24 with Glu at position 28. Alternatively, the order of theamide bond in the lactam ring can be reversed (e.g., a lactam ring canbe formed between the side chains of a Lys12 and a Glu16 oralternatively between a Glu 12 and a Lys16).

Intramolecular bridges other than a lactam bridge can be used tostabilize the alpha helix of Q. In some embodiments, the intramolecularbridge is a hydrophobic bridge. In this instance, the intramolecularbridge optionally is between the side chains of two amino acids that arepart of the hydrophobic face of the alpha helix of Q. For example, oneof the amino acids joined by the hydrophobic bridge can be the aminoacid at position 10, 14, and 18 (according to the amino acid numberingof wild type glucagon).

In one specific aspect, olefin metathesis is used to cross-link one ortwo turns of the alpha helix of Q using an all-hydrocarbon cross-linkingsystem. Q in this instance can comprise α-methylated amino acids bearingolefinic side chains of varying length and configured with either R or Sstereochemistry at the i and i+4 or i+7 positions. For example, theolefinic side can comprise (CH₂)_(n), wherein n is any integer between 1to 6. In some embodiments, n is 3 for a cross-link length of 8 atoms.Suitable methods of forming such intramolecular bridges are described inthe art. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000) and Walensky et al., Science 305: 1466-1470 (2004).Alternatively, Q can comprise O-allyl Ser residues located on adjacenthelical turns, which are bridged together via ruthenium-catalyzed ringclosing metathesis. Such procedures of cross-linking are described in,for example, Blackwell et al., Angew, Chem., Int. Ed. 37: 3281-3284(1998).

In another specific aspect, use of the unnatural thio-dialanine aminoacid, lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α,ω-diaminoalkane tethers, e.g., 1,4-diaminopropaneand 1,5-diaminopentane) between two Glu residues at positions i and i+7are used to stabilize the alpha helix of Q. Such tethers lead to theformation of a bridge of 9-atoms or more in length, depending on thelength of the diaminoalkane tether. Suitable methods of producingpeptides cross-linked with such tethers are described in the art. See,for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460 (1997).

In yet another embodiment of the invention, a disulfide bridge is usedto cross-link one or two turns of the alpha helix of Q. Alternatively, amodified disulfide bridge in which one or both sulfur atoms are replacedby a methylene group resulting in an isosteric macrocyclization is usedto stabilize the alpha helix of Q. Suitable methods of modifyingpeptides with disulfide bridges or sulfur-based cyclization aredescribed in, for example, Jackson et al., J. Am. Chem. Soc. 113:9391-9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).

In yet another embodiment, the alpha helix of Q is stabilized via thebinding of metal atom by two His residues or a His and Cys pairpositioned at i and i+4. The metal atom can be, for example, Ru(III),Cu(II), Zn(II), or Cd(II). Such methods of metal binding-based alphahelix stabilization are known in the art. See, for example, Andrews andTabor, Tetrahedron 55: 11711-11743 (1999); Ghadiri et al., J. Am. Chem.Soc. 112: 1630-1632 (1990); and Ghadiri et al., J. Am. Chem. Soc. 119:9063-9064 (1997).

The alpha helix of Q can alternatively be stabilized through other meansof peptide cyclizing, which means are reviewed in Davies, J. Peptide.Sci. 9: 471-501 (2003). The alpha helix can be stabilized via theformation of an amide bridge, thioether bridge, thioester bridge, ureabridge, carbamate bridge, sulfonamide bridge, and the like. For example,a thioester bridge can be formed between the C-terminus and the sidechain of a Cys residue. Alternatively, a thioester can be formed viaside chains of amino acids having a thiol (Cys) and a carboxylic acid(e.g., Asp, Glu). In another method, a cross-linking agent, such as adicarboxylic acid, e.g. suberic acid (octanedioic acid), etc. canintroduce a link between two functional groups of an amino acid sidechain, such as a free amino, hydroxyl, thiol group, and combinationsthereof.

In accordance with some embodiments, the alpha helix of Q is stabilizedthrough the incorporation of hydrophobic amino acids at positions i andi+4. For instance, i can be Tyr and i+4 can be either Val or Leu; i canbe Phe and i+4 can be Cys or Met; I can be Cys and i+4 can be Met; or ican be Phe and i+4 can be Ile. It should be understood that, forpurposes herein, the above amino acid pairings can be reversed, suchthat the indicated amino acid at position i could alternatively belocated at i+4, while the i+4 amino acid can be located at the iposition.

In accordance with other embodiments of the invention, wherein Q is aglucagon related peptide, the alpha helix is stabilized throughincorporation (either by amino acid substitution or insertion) of one ormore alpha helix-stabilizing amino acids at the C-terminal portion of Q(around amino acids 12-29 according to the numbering of the amino acidnumbering of wild type glucagon). In a specific embodiment, the alphahelix-stabilizing amino acid is an α,α-disubstituted amino acid,including, but not limited to any of amino iso-butyric acid (Aib), anamino acid disubstituted with the same or a different group selectedfrom methyl, ethyl, propyl, and n-butyl, or with a cyclooctane orcycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In someembodiments, one, two, three, four or more of positions 16, 17, 18, 19,20, 21, 24 or 29 of the glucagon related peptide is substituted with anα,α-disubstituted amino acid. In a specific embodiment, one, two, threeor all of positions 16, 20, 21, and 24 are substituted with Aib.

Conjugates

In some embodiments, the peptides (Q) described herein are glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into a salt (e.g., an acidaddition salt, a basic addition salt), and/or optionally dimerized,multimerized, or polymerized, or conjugated. As described herein, Q canbe a glucagon superfamily peptide, glucagon related peptide, including aClass 1, 2, 3, 4 or 5 glucagon related peptide, or osteocalcin,calcitonin, amylin, or an analog, derivative or conjugate thereof.

The present disclosure also encompasses conjugates in which Q of Q-L-Yis further linked to a heterologous moiety. The conjugation between Qand the heterologous moiety can be through covalent bonding,non-covalent bonding (e.g. electrostatic interactions, hydrogen bonds,van der Waals interactions, salt bridges, hydrophobic interactions, andthe like), or both types of bonding. A variety of non-covalent couplingsystems may be used, including biotin-avidin, ligand/receptor,enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipidbinding protein, cellular adhesion molecule partners; or any bindingpartners or fragments thereof which have affinity for each other. Insome aspects, the covalent bonds are peptide bonds. The conjugation of Qto the heterologous moiety may be indirect or direct conjugation, theformer of which may involve a linker or spacer. Suitable linkers andspacers are known in the art and include, but not limited to, any of thelinkers or spacers described herein under the sections “Acylation andalkylation” and “The Linking Group,” and in the subsection “ChemicalModification of Q and/or L.”

As used herein, the term “heterologous moiety” is synonymous with theterm “conjugate moiety” and refers to any molecule (chemical orbiochemical, naturally-occurring or non-coded) which is different from Qto which it is attached. Exemplary conjugate moieties that can be linkedto Q include but are not limited to a heterologous peptide orpolypeptide (including for example, a plasma protein), a targetingagent, an immunoglobulin or portion thereof (e.g., variable region, CDR,or Fc region), a diagnostic label such as a radioisotope, fluorophore orenzymatic label, a polymer including water soluble polymers, or othertherapeutic or diagnostic agents. In some embodiments a conjugate isprovided comprising Q and a plasma protein, wherein the plasma proteinis selected from the group consisting of albumin, transferin, fibrinogenand globulins. In some embodiments the plasma protein moiety of theconjugate is albumin or transferin. The conjugate in some embodimentscomprises Q and one or more of a polypeptide, a nucleic acid molecule,an antibody or fragment thereof, a polymer, a quantum dot, a smallmolecule (molecular weight less than 1000 daltons), a toxin, adiagnostic agent, a carbohydrate, an amino acid.

C-Terminal Heterologous Moiety

In some embodiments, the heterologous moiety conjugated to Q is apeptide which is distinct from Q and the conjugate is a fusion peptideor a chimeric peptide. In some embodiments, where Q is a glucagonsuperfamily peptide, the heterologous moiety is a peptide extension of1-21 amino acids. In specific embodiments, where Q is a glucagon relatedpeptide (e.g. a Class 1, 2, 3, 4 or 5 glucagon related peptide), theextension is attached to the C-terminus of Q, e.g., to amino acid atposition 29. In some embodiments, the extension comprises an amino acidsequence of SEQ ID NO: 1610 (GPSSGAPPPS), SEQ ID NO: 1611(GGPSSGAPPPS-CONH₂), SEQ ID NO: 1614 (KRNRNNIA), SEQ ID NO: 1643 (KRNR),or KGKKNDWKHNITQ (SEQ ID NO: 1613). In specific aspects, the amino acidsequence is attached through the C-terminal amino acid of Q, e.g., aminoacid at position 29. In some embodiments, the amino acid sequence of SEQID NOs: 1610, 1611, 1613, 1614 and 1643 is bound to amino acid 29 of thepeptide through a peptide bond. In some specific embodiments, the aminoacid at position 29 of the glucagon related peptide (e.g. a Class 1, 2,3, 4 or 5 glucagon related peptide) is a Gly and the Gly is fused to oneof the amino acid sequences of SEQ ID NOs: 1610, 1611, 1613, 1614 and1643.

Polymer Heterologous Moiety

In some embodiments, the heterologous moiety conjugated to Q is apolymer. In some embodiments, the polymer is selected from the groupconsisting of: polyamides, polycarbonates, polyalkylenes and derivativesthereof including, polyalkylene glycols, polyalkylene oxides,polyalkylene terepthalates, polymers of acrylic and methacrylic esters,including poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Hydrophilic polymers are further described hereinunder “Hydrophilic Heterologous Moieties.” Suitable water-solublepolymers are known in the art and include, for example,polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel),hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose,hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose,hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose(Ethocel), hydroxyethyl cellulose, various alkyl celluloses andhydroxyalkyl celluloses, various cellulose ethers, cellulose acetate,carboxymethyl cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, vinyl acetate/crotonic acid copolymers,poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylicacid copolymers, polymethacrylic acid, polymethylmethacrylate, maleicanhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium andcalcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers,carboxypolymethylene, carboxyvinyl polymers, polyoxyethylenepolyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,carboxymethylamide, potassium methacrylate divinylbenzene co-polymer,polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, andcombinations thereof.

In specific embodiments, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

Fc Fusion Heterologous Moiety

As noted above, in some embodiments Q is conjugated, e.g., fused to animmunoglobulin or portion thereof (e.g. variable region, CDR, or Fcregion). As described herein, Q can be a glucagon superfamily peptide,glucagon related peptide, including a Class 1, 2, 3, 4 or 5 glucagonrelated peptide, or osteocalcin, calcitonin, amylin, or an analog,derivative or conjugate thereof. Known types of immunoglobulins (Ig)include IgG, IgA, IgE, IgD or IgM. The Fc region is a C-terminal regionof an Ig heavy chain, which is responsible for binding to Fc receptorsthat carry out activities such as recycling (which results in prolongedhalf-life), antibody dependent cell-mediated cytotoxicity (ADCC), andcomplement dependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a related embodiment,the Fc region may comprise one or more native or modified constantregions from an immunoglobulin heavy chain, other than CH1, for example,the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions ofIgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J Immunol. 175:6694-701, 2005) Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591) Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Hydrophilic Heterologous Moiety

In some embodiments, Q described herein is covalently bonded to ahydrophilic moiety. As described herein, Q can be a glucagon superfamilypeptide, glucagon related peptide, including a Class 1, 2, 3, 4 or 5glucagon related peptide, or osteocalcin, calcitonin, amylin, or ananalog, derivative or conjugate thereof. Hydrophilic moieties can beattached to Q under any suitable conditions used to react a protein withan activated polymer molecule. Any means known in the art can be used,including via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the PEG moiety (e.g., an aldehyde, amino, ester,thiol, α-haloacetyl, maleimido or hydrazino group) to a reactive groupon the target compound (e.g., an aldehyde, amino, ester, thiol,α-haloacetyl, maleimido or hydrazino group). Activating groups which canbe used to link the water soluble polymer to one or more proteinsinclude without limitation sulfone, maleimide, sulfhydryl, thiol,triflate, tresylate, azidirine, oxirane, 5-pyridyl, andalpha-halogenated acyl group (e.g., alpha-iodo acetic acid,alpha-bromoacetic acid, alpha-chloroacetic acid). If attached to thepeptide by reductive alkylation, the polymer selected should have asingle reactive aldehyde so that the degree of polymerization iscontrolled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev.54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476(2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

Further activating groups which can be used to link the hydrophilicmoiety (water soluble polymer) to a protein include an alpha-halogenatedacyl group (e.g., alpha-iodo acetic acid, alpha-bromoacetic acid,alpha-chloroacetic acid). In specific aspects, an amino acid residue ofthe peptide having a thiol is modified with a hydrophilic moiety such asPEG. In some embodiments, an amino acid on Q comprising a thiol ismodified with maleimide-activated PEG in a Michael addition reaction toresult in a PEGylated peptide comprising the thioether linkage shownbelow:

In some embodiments, the thiol of an amino acid of Q is modified with ahaloacetyl-activated PEG in a nucleophilic substitution reaction toresult in a PEGylated peptide comprising the thioether linkage shownbelow:

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.Dextrans are polysaccharide polymers of glucose subunits, predominantlylinked by al-6 linkages. Dextran is available in many molecular weightranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD.

The hydrophilic moiety, e.g., polyethylene glycol chain, in accordancewith some embodiments has a molecular weight selected from the range ofabout 500 to about 40,000 Daltons. In some embodiments the polyethyleneglycol chain has a molecular weight selected from the range of about 500to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons. Inanother embodiment the hydrophilic moiety, e.g., polyethylene glycolchain, has a molecular weight of about 10,000 to about 20,000 Daltons.In yet other exemplary embodiments the hydrophilic moiety, e.g.polyethylene glycol chain, has a molecular weight of about 20,000 toabout 40,000 Daltons.

Linear or branched hydrophilic polymers are contemplated. Resultingpreparations of conjugates may be essentially monodisperse orpolydisperse, and may have about 0.5, 0.7, 1, 1.2, 1.5 or 2 polymermoieties per peptide.

In some embodiments, the native amino acid of the peptide is substitutedwith an amino acid having a side chain suitable for crosslinking withhydrophilic moieties, to facilitate linkage of the hydrophilic moiety tothe peptide. Exemplary amino acids include Cys, Lys, Orn, homo-Cys, oracetyl phenylalanine (Ac-Phe). In other embodiments, an amino acidmodified to comprise a hydrophilic group is added to the peptide at theC-terminus

In some embodiments, the peptide of the conjugate is conjugated to ahydrophilic moiety, e.g. PEG, via covalent linkage between a side chainof an amino acid of the peptide and the hydrophilic moiety. In someembodiments, where Q is a Class 1, 2, 3, 4 or 5 glucagon-relatedpeptide, the peptide is conjugated to a hydrophilic moiety via the sidechain of an amino acid at position 16, 17, 21, 24, 29, 40, a positionwithin a C-terminal extension, or the C-terminal amino acid, or acombination of these positions. In some aspects, the amino acidcovalently linked to a hydrophilic moiety (e.g., the amino acidcomprising a hydrophilic moiety) is a Cys, Lys, Orn, homo-Cys, orAc-Phe, and the side chain of the amino acid is covalently bonded to ahydrophilic moiety (e.g., PEG).

rPEG Heterologous Moiety

In some embodiments, the conjugate of the invention comprises a Q fusedto an accessory peptide which is capable of forming an extendedconformation similar to chemical PEG (e.g., a recombinant PEG (rPEG)molecule), such as those described in International Patent ApplicationPublication No. WO2009/023270 and U.S. Patent Application PublicationNo. US2008/0286808. The rPEG molecule is not polyethylene glycol. TherPEG molecule in some aspects is a polypeptide comprising one or more ofglycine, serine, glutamic acid, aspartic acid, alanine, or proline. Insome aspects, the rPEG is a homopolymer, e.g., poly-glycine,poly-serine, poly-glutamic acid, poly-aspartic acid, poly-alanine, orpoly-proline. In other embodiments, the rPEG comprises two types ofamino acids repeated, e.g., poly(Gly-Ser), poly(Gly-Glu), poly(Gly-Ala),poly(Gly-Asp), poly(Gly-Pro), poly(Ser-Glu), etc. In some aspects, therPEG comprises three different types of amino acids, e.g.,poly(Gly-Ser-Glu). In specific aspects, the rPEG increases the half-lifeof Q. In some aspects, the rPEG comprises a net positive or net negativecharge. The rPEG in some aspects lacks secondary structure. In someembodiments, the rPEG is greater than or equal to 10 amino acids inlength and in some embodiments is about 40 to about 50 amino acids inlength. The accessory peptide in some aspects is fused to the N- orC-terminus of the peptide of the invention through a peptide bond or aproteinase cleavage site, or is inserted into the loops of the peptideof the invention. The rPEG in some aspects comprises an affinity tag oris linked to a PEG that is greater than 5 kDa. In some embodiments, therPEG confers the conjugate of the invention with an increasedhydrodynamic radius, serum half-life, protease resistance, or solubilityand in some aspects confers the conjugate with decreased immunogenicity.

Q can be linked to conjugate moieties via direct covalent linkage byreacting targeted amino acid residues of the peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of these targeted amino acids. Reactivegroups on the peptide or conjugate moiety include, e.g., an aldehyde,amino, ester, thiol, α-haloacetyl, maleimido or hydrazino group.Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art. Alternatively, the conjugatemoieties can be linked to the peptide indirectly through intermediatecarriers, such as polysaccharide or polypeptide carriers. Examples ofpolysaccharide carriers include aminodextran. Examples of suitablepolypeptide carriers include polylysine, polyglutamic acid, polyasparticacid, co-polymers thereof, and mixed polymers of these amino acids andothers, e.g., serines, to confer desirable solubility properties on theresultant loaded carrier.

Multimers

With regard to the Class 1, Class 2, and Class 3 glucagon relatedpeptides, Q may be part of a dimer, trimer or higher order multimercomprising at least two, three, or more peptides bound via a linker,wherein at least one or both peptides is a glucagon related peptide. Thedimer may be a homodimer or heterodimer. In some embodiments, the linkeris selected from the group consisting of a bifunctional thiolcrosslinker and a bi-functional amine crosslinker. In certainembodiments, the linker is PEG, e.g., a 5 kDa PEG, 20 kDa PEG. In someembodiments, the linker is a disulfide bond. For example, each monomerof the dimer may comprise a Cys residue (e.g., a terminal or internallypositioned Cys) and the sulfur atom of each Cys residue participates inthe formation of the disulfide bond. In some aspects of the invention,the monomers are connected via terminal amino acids (e.g., N-terminal orC-terminal), via internal amino acids, or via a terminal amino acid ofat least one monomer and an internal amino acid of at least one othermonomer. In specific aspects, the monomers are not connected via anN-terminal amino acid. In some aspects, the monomers of the multimer areattached together in a “tail-to-tail” orientation in which theC-terminal amino acids of each monomer are attached together. Aconjugate moiety may be covalently linked to any of the glucagon relatedpeptides described herein, including a dimer, trimer or higher ordermultimer.

Conjugation of the Heterologous Moiety to Q

The heterologous moiety is conjugated to peptide (Q) according tolinkage and conjugation methods described in the “Linking Group” sectionand “Chemical Modification of Q and/or Y subsection.”

Methods for Making Q

The peptides (Q) disclosed herein may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

The peptides of the disclosure can be obtained by methods known in theart. Suitable methods of de novo synthesizing peptides are described in,for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, OxfordUniversity Press, Oxford, United Kingdom, 2005; Peptide and Protein DrugAnalysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed.Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000;and U.S. Pat. No. 5,449,752.

Also, in the instances in which the peptides of the disclosure do notcomprise any non-coded or non-natural amino acids, the peptide can berecombinantly produced using a nucleic acid encoding the amino acidsequence of the peptide using standard recombinant methods. See, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994.

In some embodiments, the peptides of the disclosure are isolated. Insome embodiments, the peptides of the disclosure are purified. It isrecognized that “purity” is a relative term, and not to be necessarilyconstrued as absolute purity or absolute enrichment or absoluteselection. In some aspects, the purity is at least or about 50%, is atleast or about 60%, at least or about 70%, at least or about 80%, or atleast or about 90% (e.g., at least or about 91%, at least or about 92%,at least or about 93%, at least or about 94%, at least or about 95%, atleast or about 96%, at least or about 97%, at least or about 98%, atleast or about 99% or is approximately 100%.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

Classes of glucagon related peptides (Q) are described in detail below.With respect to each of the sections of disclosure concerning Class 1,Class 2, Class 3, Class 4, and Class 5 glucagon related peptides,modifications are described with respect to the glucagon related peptideportion (Q) of a Q-L-Y conjugate detailed above. Thus, structuralelements described with regard to a class of glucagon related peptidesare structural elements of Q which is then further modified to generatethe Q-L-Y conjugate as described above.

Class 1 Glucagon Related Peptides

In certain embodiments, the glucagon related peptide is a Class 1glucagon related peptide, which is described herein and in InternationalPatent Publication No. WO 2009/155257 (published on Dec. 23, 2009),International Patent Application Publication No. WO 2008/086086(published on Jul. 17, 2008), and International Patent ApplicationPublication No. WO 2007/056362 (published on May 18, 2007), the contentsof which are incorporated by reference in their entirety.

The biological sequences referenced in the following section (SEQ IDNOs: 801-915) relating to Class 1 glucagon related peptides correspondto SEQ ID NOs: 1-115 in International International Patent PublicationNo. WO 2009/155257.

Activity

Class 1 glucagon peptides retain glucagon receptor activity relative tothe native glucagon peptide (SEQ ID NO: 801). For example, the glucagonpeptide can retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%activity, 80% activity, 85% activity, or 90% of the activity of nativeglucagon (calculated as the inverse ratio of EC₅₀s for the glucagonpeptide vs. glucagon, e.g., as measured by cAMP production using theassay generally described in Example 2). In some embodiments, the Class1 glucagon related peptides have the same or greater activity (usedsynonymously with the term “potency” herein) than glucagon. In someembodiments, the glucagon peptides described herein exhibit no more thanabout 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity ofnative glucagon peptide.

Any of the Class 1 glucagon related peptides described herein mayexhibit an EC₅₀ at the human glucagon receptor of about 100 nM, 75 nM,50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM or less when tested forcAMP induction in HEK293 cells over-expressing glucagon receptor, e.g.using the assay of Example 2. Typically pegylated peptides will exhibita higher EC₅₀ compared to the unpegylated peptide. For example, theClass 1 glucagon related peptides described herein, when unpegylated,may exhibit activity at the glucagon receptor which is at least 20%(e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least75%, at least 80%, at least 90% at least 95%, at least 98%, at least99%, 100%, 150%, 200%, 400%, 500% or more) of the activity of nativeglucagon (SEQ ID NO: 801) at the glucagon receptor. In certainembodiments, the Class 1 glucagon related peptides described hereinexhibit the indicated % activity of native glucagon at the glucagonreceptor, when lacking a hydrophilic moiety, but exhibit a decreased %activity of native glucagon at the glucagon receptor, when comprising ahydrophilic moiety. For example, the Class 1 glucagon related peptidesdescribed herein, when pegylated, may exhibit activity at the glucagonreceptor which is at least 2% (e.g. at least 3%, at least 4%, at least5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%of the activity of native glucagon. In some embodiments, the Class 1glucagon related peptides described herein may exhibit any of the aboveindicated activities but no more than 1000%, 10,000%, 100,000%, or1,000,000% of the activity of native glucagon at the glucagon receptor.

In some embodiments, the Class 1 glucagon related peptides exhibit lessthan about 5%, 4%, 3%, 2% or 1% of the activity of native GLP-1 at theGLP-1 receptor and/or a greater than about 5-fold, 10-fold, or 15-foldselectivity for glucagon receptor compared to GLP-1 receptor. Forexample, in some embodiments, the Class 1 glucagon related peptidesexhibit less than 5% of the activity of native GLP-1 at the GLP-1receptor and exhibit a greater than 5-fold selectivity for glucagonreceptor compared to GLP-1 receptor.

Improved Solubility

Native glucagon exhibits poor solubility in aqueous solution,particularly at physiological pH, with a tendency to aggregate andprecipitate over time. In contrast, the Class 1 glucagon relatedpeptides in some embodiments exhibit at least 2-fold, 5-fold, or evenhigher solubility compared to native glucagon at a pH between 6 and 8,or between 6 and 9, for example, at pH 7 after 24 hours at 25° C.

Accordingly, in some embodiments, a Class 1 glucagon related peptide hasbeen modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 801) to improve the peptides solubility in aqueoussolutions, particularly at a pH ranging from about 5.5 to about 8.0,while retaining the native peptide's biological activity.

For example, the solubility of any of the Class 1 glucagon relatedpeptides described herein can be further improved by attaching ahydrophilic moiety to the peptide. Introduction of such groups alsoincreases duration of action, e.g. as measured by a prolonged half-lifein circulation. Hydrophilic moieties are further described herein.

Modification with Charged Residues

In some embodiments, solubility is improved by adding charge to theClass 1 glucagon related peptide by the substitution of nativenon-charged amino acids with charged amino acids selected from the groupconsisting of lysine, arginine, histidine, aspartic acid and glutamicacid, or by the addition of charged amino acids to the amino or carboxyterminus of the peptide.

In accordance with some embodiments, the Class 1 glucagon relatedpeptide has improved solubility due to the fact that the peptide ismodified by amino acid substitutions and/or additions that introduce acharged amino acid into the C-terminal portion of the peptide, and insome embodiments at a position C-terminal to position 27 of SEQ ID NO:801. Optionally, one, two or three charged amino acids may be introducedwithin the C-terminal portion, and in some embodiments C-terminal toposition 27. In accordance with some embodiments, the native aminoacid(s) at positions 28 and/or 29 are substituted with a charged aminoacid, and/or one to three charged amino acids are added to theC-terminus of the peptide, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged.

In specific exemplary embodiments, the Class 1 glucagon related peptidemay comprise any one or two of the following modifications: substitutionof N28 with E; substitution of N28 with D; substitution of T29 with D;substitution of T29 with E; insertion of E after position 27, 28 or 29;insertion of D after position 27, 28 or 29. For example, D28E29, E28E29,E29E30, E28E30, D28E30.

In accordance with one exemplary embodiment, the Class 1 glucagonrelated peptide comprises an amino acid sequence of SEQ ID NO: 811, oran analog thereof that contains 1 to 3 further amino acid modifications(described herein in reference to glucagon agonists) relative to nativeglucagon, or a glucagon agonist analog thereof. SEQ ID NO: 811represents a modified Class 1 glucagon related peptide, wherein theasparagine residue at position 28 of the native protein has beensubstituted with an aspartic acid. In another exemplary embodiment theClass 1 glucagon related peptide comprises an amino acid sequence of SEQID NO: 838, wherein the asparagine residue at position 28 of the nativeprotein has been substituted with glutamic acid. Other exemplaryembodiments include Class 1 glucagon related peptides of SEQ ID NOs:824, 825, 826, 833, 835, 836 and 837.

Substituting the normally occurring amino acid at position 28 and/or 29with charged amino acids, and/or the addition of one to two chargedamino acids at the carboxy terminus of the Class 1 glucagon relatedpeptide, enhances the solubility and stability of the glucagon peptidesin aqueous solutions at physiologically relevant pHs (i.e., a pH ofabout 6.5 to about 7.5) by at least 5-fold and by as much as 30-fold.Accordingly, Class 1 glucagon peptides of some embodiments retainglucagon activity and exhibit at least 2-fold, 5-fold, 10-fold, 15-fold,25-fold, 30-fold or greater solubility relative to native glucagon at agiven pH between about 5.5 and 8, e.g., pH 7, when measured after 24hours at 25° C.

Additional modifications, e.g. conservative substitutions, whichmodifications are further described herein, may be made to the Class 1glucagon related peptide that still allow it to retain glucagonactivity.

Improved Stability

Any of the Class 1 glucagon peptides may additionally exhibit improvedstability and/or reduced degradation, for example, retaining at least95% of the original peptide after 24 hours at 25° C. Any of the Class 1glucagon related peptides disclosed herein may additionally exhibitimproved stability at a pH within the range of 5.5 to 8, for example,retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of theoriginal peptide after 24 hours at 25° C. In some embodiments, the Class1 glucagon related peptides of the invention exhibit improved stability,such that at least 75% (e.g., at least 80%, at least 85%, at least 90%,at least 95%, more than 95%, up to 100%) of a concentration of thepeptide or less than about 25% (e.g., less than 20%, less than 15%, lessthan 10%, less than 5%, 4%, 3%, 2%, 1%, down to 0%) of degraded peptideis detectable at 280 nm by an ultraviolet (UV) detector after about 1 ormore weeks (e.g., about 2 weeks, about 4 weeks, about 1 month, about twomonths, about four months, about six months, about eight months, aboutten months, about twelve months) in solution at a temperature of atleast 20° C. (e.g., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., atleast 27.5° C., at least 30° C., at least 35° C., at least 40° C., atleast 50° C.) and less than 100° C., less than 85° C., less than 75° C.,or less than 70° C. The Class 1 glucagon related peptides may includeadditional modifications that alter its pharmaceutical properties, e.g.increased potency, prolonged half-life in circulation, increasedshelf-life, reduced precipitation or aggregation, and/or reduceddegradation, e.g., reduced occurrence of cleavage or chemicalmodification after storage.

In yet further exemplary embodiments, any of the foregoing Class 1glucagon related peptides can be further modified to improve stabilityby modifying the amino acid at position 15 of SEQ ID NO: 801 to reducedegradation of the peptide over time, especially in acidic or alkalinebuffers. In exemplary embodiments, Asp at position 15 is substitutedwith a Glu, homo-Glu, cysteic acid, or homo-cysteic acid.

Alternatively, any of the Class 1 glucagon related peptides describedherein can be further modified to improve stability by modifying theamino acid at position 16 of SEQ ID NO: 801. In exemplary embodiments,Ser at position 16 is substituted with Thr or Aib, or any of the aminoacids substitutions described herein with regard to Class 1 glucagonrelated peptides which enhance potency at the glucagon receptor. Suchmodifications reduce cleavage of the peptide bond between Asp15-Ser16.

In some embodiments, any of the Class 1 glucagon related peptidesdescribed herein can be further modified to reduce degradation atvarious amino acid positions by modifying any one, two, three, or allfour of positions 20, 21, 24, or 27. Exemplary embodiments includesubstitution of Gln at position 20 with Ser, Thr, Ala or Aib,substitution of Asp at position 21 with Glu, substitution of Gln atposition 24 with Ala or Aib, substitution of Met at position 27 with Leuor Nle. Removal or substitution of methionine reduces degradation due tooxidation of the methionine. Removal or substitution of Gln or Asnreduces degradation due to deamidation of Gln or Asn. Removal orsubstitution of Asp reduces degradation that occurs through dehydrationof Asp to form a cyclic succinimide intermediate followed byisomerization to iso-aspartate.

Enhanced Potency

In accordance with another embodiment, Class 1 glucagon related peptidesare provided that have enhanced potency at the glucagon receptor,wherein the peptides comprise an amino acid modification at position 16of native glucagon (SEQ ID NO: 801). By way of nonlimiting example, suchenhanced potency can be provided by substituting the naturally occurringserine at position 16 with glutamic acid or with another negativelycharged amino acid having a side chain with a length of 4 atoms, oralternatively with any one of glutamine, homoglutamic acid, orhomocysteic acid, or a charged amino acid having a side chain containingat least one heteroatom, (e.g. N, O, S, P) and with a side chain lengthof about 4 (or 3-5) atoms. Substitution of serine at position 16 withglutamic acid enhances glucagon activity at least 2-fold, 4-fold, 5-foldand up to 10-fold greater at the glucagon receptor. In some embodiments,the Class 1 glucagon related peptide retains selectivity for theglucagon receptor relative to the GLP-1 receptors, e.g., at least5-fold, 10-fold, or 15-fold selectivity.

DPP-IV Resistance

In some embodiments, the Class 1 glucagon peptides disclosed herein arefurther modified at position 1 or 2 to reduce susceptibility to cleavageby dipeptidyl peptidase IV. More particularly, in some embodiments,position 1 and/or position 2 of the Class 1 glucagon related peptide issubstituted with the DPP-IV resistant amino acid(s) described herein. Insome embodiments, position 2 of the analog peptide is substituted withan amino isobutyric acid. In some embodiments, position 2 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of D-serine, D-alanine, glycine, N-methyl serine, and ε-aminobutyric acid. In another embodiment, position 2 of the Class 1 glucagonrelated peptide is substituted with an amino acid selected from thegroup consisting of D-serine, glycine, and aminoisobutyric acid. In someembodiments, the amino acid at position 2 is not D-serine.

Reduction in glucagon activity upon modification of the amino acids atposition 1 and/or position 2 of the glucagon peptide can be restored bystabilization of the alpha-helix structure in the C-terminal portion ofthe glucagon peptide (around amino acids 12-29). The alpha helixstructure can be stabilized by, e.g., formation of a covalent ornon-covalent intramolecular bridge (e.g., a lactam bridge between sidechains of amino acids at positions “i” and “i+4”, wherein i is aninteger from 12 to 25), substitution and/or insertion of amino acidsaround positions 12-29 with an alpha helix-stabilizing amino acid (e.g.,an α,α-disubstituted amino acid), as further described herein.

Modifications at Position 3

Glucagon receptor activity can be reduced by an amino acid modificationat position 3 (according to the amino acid numbering of wild typeglucagon), e.g. substitution of the naturally occurring glutamine atposition 3, with an acidic, basic, or a hydrophobic amino acid. Forexample substitution at position 3 with glutamic acid, ornithine, ornorleucine substantially reduces or destroys glucagon receptor activity.

Maintained or enhanced activity at the glucagon receptor may be achievedby modifying the Gln at position 3 with a glutamine analog as describedherein. For example, glucagon agonists can comprise the amino acidsequence of SEQ ID NO: 863, SEQ ID NO: 869, SEQ ID NO: 870, SEQ ID NO:871, SEQ ID NO: 872, SEQ ID NO: 873, and SEQ ID NO: 874.

Enhancing GLP-1 Activity with C-Terminal Amides and Esters

Enhanced activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester. Conversely, retaining the nativecarboxylic acid at the C-terminus of the peptide maintains therelatively greater selectivity of the Class 1 glucagon related peptidefor the glucagon receptor vs. the GLP-1 receptor (e.g., greater thanabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20-fold).

Further Modifications and Combinations

Additional modifications may be made to the Class 1 glucagon relatedpeptide which may further increase solubility and/or stability and/orglucagon activity. The Class 1 glucagon related peptide mayalternatively comprise other modifications that do not substantiallyaffect solubility or stability, and that do not substantially decreaseglucagon activity. In exemplary embodiments, the Class 1 glucagonrelated peptide may comprise a total of up to 11, or up to 12, or up to13, or up to 14 amino acid modifications relative to the native glucagonsequence. For example, conservative or non-conservative substitutions,additions or deletions may be carried out at any of positions 2, 5, 7,10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29.

Exemplary modifications of the Class 1 glucagon related peptide includebut are not limited to:

(a) non-conservative substitutions, conservative substitutions,additions or deletions while retaining at least partial glucagon agonistactivity, for example, conservative substitutions at one or more ofpositions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27,28 or 29, substitution of Tyr at position 10 with Val or Phe,substitution of Lys at position 12 with Arg, substitution of one or moreof these positions with Ala;

(b) deletion of amino acids at positions 29 and/or 28, and optionallyposition 27, while retaining at least partial glucagon agonist activity;

(c) modification of the aspartic acid at position 15, for example, bysubstitution with glutamic acid, homoglutamic acid, cysteic acid orhomocysteic acid, which may reduce degradation; or modification of theserine at position 16, for example, by substitution of threonine, Aib,glutamic acid or with another negatively charged amino acid having aside chain with a length of 4 atoms, or alternatively with any one ofglutamine, homoglutamic acid, or homocysteic acid, which likewise mayreduce degradation due to cleavage of the Asp15-Ser16 bond;

(d) addition of a hydrophilic moiety such as the water soluble polymerpolyethylene glycol, as described herein, e.g. at position 16, 17, 20,21, 24, 29, 40 or at the C-terminal amino acid, which may increasesolubility and/or half-life;

(e) modification of the methionine at position 27, for example, bysubstitution with leucine or norleucine, to reduce oxidativedegradation;

(f) modification of the Gln at position 20 or 24, e.g. by substitutionwith Ser, Thr, Ala or Aib, to reduce degradation that occurs throughdeamidation of Gln

(g) modification of Asp at position 21, e.g. by substitution with Glu,to reduce degradation that occurs through dehydration of Asp to form acyclic succinimide intermediate followed by isomerization toiso-aspartate;

(h) modifications at position 1 or 2 as described herein that improveresistance to DPP-IV cleavage, optionally in combination with anintramolecular bridge such as a lactam bridge between positions “i” and“i+4”, wherein i is an integer from 12 to 25, e.g., 12, 16, 20, 24;

(i) acylating or alkylating the glucagon peptide as described herein,which may increase the activity at the glucagon receptor and/or theGLP-1 receptor, increase half-life in circulation and/or extending theduration of action and/or delaying the onset of action, optionallycombined with addition of a hydrophilic moiety, additionally oralternatively, optionally combined with a modification which selectivelyreduces activity at the GLP-1 peptide, e.g., a modification of the Thrat position 7, such as a substitution of the Thr at position 7 with anamino acid lacking a hydroxyl group, e.g., Abu or Ile; deleting aminoacids C-terminal to the amino acid at position 27 (e.g., deleting one orboth of the amino acids at positions 28 and 29, yielding a peptide 27 or28 amino acids in length);

(j)C-terminal extensions as described herein;

(k) homodimerization or heterodimerization as described herein; and

combinations of the (a) through (k).

In some embodiments, exemplary modifications of the Class 1 glucagonrelated peptide include at least one amino acid modification selectedfrom Group A and one or more amino acid modifications selected fromGroup B and/or Group C,

wherein Group A is:

substitution of Asn at position 28 with a charged amino acid;

substitution of Asn at position 28 with a charged amino acid selectedfrom the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, andhomocysteic acid;

substitution at position 28 with Asn, Asp, or Glu;

substitution at position 28 with Asp;

substitution at position 28 with Glu;

substitution of Thr at position 29 with a charged amino acid;

substitution of Thr at position 29 with a charged amino acid selectedfrom the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, andhomocysteic acid;

substitution at position 29 with Asp, Glu, or Lys;

substitution at position 29 with Glu;

insertion of 1-3 charged amino acids after position 29;

insertion after position 29 of Glu or Lys;

insertion after position 29 of Gly-Lys or Lys-Lys;

or combinations thereof;

wherein Group B is:

substitution of Asp at position 15 with Glu;

substitution of Ser at position 16 with Thr or Aib;

and wherein Group C is:

substitution of His at position 1 with a non-native amino acid thatreduces susceptibility of the glucagon peptide to cleavage by dipeptidylpeptidase IV (DPP-IV), substitution of Ser at position 2 with anon-native amino acid that reduces susceptibility of the glucagonpeptide to cleavage by dipeptidyl peptidase IV (DPP-IV), substitution ofLys at position 12 with Arg;

substitution of Gln at position 20 with Ser, Thr, Ala or Aib;

substitution of Asp at position 21 with Glu;

substitution of Gln at position 24 with Ser, Thr, Ala or Aib;

substitution of Met at position 27 with Leu or Nle;

deletion of amino acids at positions 27-29;

deletion of amino acids at positions 28-29;

deletion of the amino acid at positions 29;

or combinations thereof.

In exemplary embodiments, Lys at position 12 is substituted with Arg. Inother exemplary embodiments amino acids at positions 29 and/or 28, andoptionally at position 27, are deleted.

In some specific embodiments, the glucagon peptide comprises (a) anamino acid modification at position 1 and/or 2 that confers DPP-IVresistance, e.g., substitution with DMIA at position 1, or Aib atposition 2, (b) an intramolecular bridge within positions 12-29, e.g. atpositions 16 and 20, or one or more substitutions of the amino acids atpositions 16, 20, 21, and 24 with an α,α disubstituted amino acid,optionally (c) linked to a hydrophilic moiety such as PEG, e.g., throughCys at position 24, 29 or at the C-terminal amino acid, optionally (d)an amino acid modification at position 27 that substitutes Met with,e.g., Nle, optionally (e) amino acid modifications at positions 20, 21and 24 that reduce degradation, and optionally (f) linked to SEQ ID NO:820. When the glucagon peptide is linked to SEQ ID NO: 820, the aminoacid at position 29 in certain embodiments is Thr or Gly. In otherspecific embodiments, the glucagon peptide comprises (a) Asp28Glu29, orGlu28Glu29, or Glu29Glu30, or Glu28Glu30 or Asp28Glu30, and optionally(b) an amino acid modification at position 16 that substitutes Ser with,e.g. Thr or Aib, and optionally (c) an amino acid modification atposition 27 that substitutes Met with, e.g., Nle, and optionally (d)amino acid modifications at positions 20, 21 and 24 that reducedegradation. In a specific embodiment, the glucagon peptide is T16, A20,E21, A24, Nle27, D28, E29.

In some embodiments, the Class 1 glucagon related peptide comprises theamino acid sequence:

X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z(SEQ ID NO: 839) with 1 to 3 amino acid modifications thereto,

wherein X1 and/or X2 is a non-native amino acid that reducessusceptibility of (or increases resistance of) the glucagon peptide tocleavage by dipeptidyl peptidase IV (DPP-IV),

wherein Z is selected from the group consisting of —COOH (the naturallyoccurring C-terminal carboxylate), -Asn-COOH, Asn-Thr-COOH, and Y—COOH,wherein Y is 1 to 2 amino acids, and

wherein an intramolecular bridge, preferably a covalent bond, connectsthe side chains of an amino acid at position i and an amino acid atposition i+4, wherein i is 12, 16, 20 or 24.

In some embodiments, the intramolecular bridge is a lactam bridge. Insome embodiments, the amino acids at positions i and i+4 of SEQ ID NO:839 are Lys and Glu, e.g., Glu16 and Lys20. In some embodiments, X1 isselected from the group consisting of: D-His, N-methyl-His,alpha-methyl-His, imidazole acetic acid, des-amino-His, hydroxyl-His,acetyl-His, homo-His, and alpha, alpha-dimethyl imidiazole acetic acid(DMIA). In other embodiments, X2 is selected from the group consistingof: D-Ser, D-Ala, Gly, N-methyl-Ser, Val, and alpha, amino isobutyricacid (Aib). In some embodiments, the glucagon peptide is covalentlylinked to a hydrophilic moiety at any of amino acid positions 16, 17,20, 21, 24, 29, 40, within a C-terminal extension, or at the C-terminalamino acid. In exemplary embodiments, this hydrophilic moiety iscovalently linked to a Lys, Cys, Orn, homocysteine, oracetyl-phenylalanine residue at any of these positions. Exemplaryhydrophilic moieties include polyethylene glycol (PEG), for example, ofa molecular weight of about 1,000 Daltons to about 40,000 Daltons, orabout 20,000 Daltons to about 40,000 Daltons.

In other embodiments, the Class I glucagon related peptide comprises theamino acid sequence:

(SEQ ID NO: 839) X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln- Trp-Leu-Met-Z,

wherein X1 and/or X2 is a non-native amino acid that reducessusceptibility of (or increases resistance of) the glucagon peptide tocleavage by dipeptidyl peptidase IV (DPP-IV),

wherein one, two, three, four or more of positions 16, 20, 21, and 24 ofthe glucagon peptide is substituted with an α,α-disubstituted aminoacid, and

wherein Z is selected from the group consisting of —COOH (the naturallyoccurring C-terminal carboxylate), -Asn-COOH, Asn-Thr-COOH, and Y—COOH,wherein Y is 1 to 2 amino acids.

Exemplary further amino acid modifications to the foregoing Class 1glucagon related peptides or analogs include substitution of Thr atposition 7 with an amino acid lacking a hydroxyl group, e.g.,aminobutyric acid (Abu), Ile, optionally, in combination withsubstitution or addition of an amino acid comprising a side chaincovalently attached (optionally, through a spacer) to an acyl or alkylgroup, which acyl or alkyl group is non-native to a naturally-occurringamino acid, substitution of Lys at position 12 with Arg; substitution ofAsp at position 15 with Glu; substitution of Ser at position 16 with Thror Aib; substitution of Gln at position 20 with Ser, Thr, Ala or Aib;substitution of Asp at position 21 with Glu; substitution of Gln atposition 24 with Ser, Thr, Ala or Aib; substitution of Met at position27 with Leu or Nle; substitution of Asn at position 28 with a chargedamino acid; substitution of Asn at position 28 with a charged amino acidselected from the group consisting of Lys, Arg, His, Asp, Glu, cysteicacid, and homocysteic acid; substitution at position 28 with Asn, Asp,or Glu; substitution at position 28 with Asp; substitution at position28 with Glu; substitution of Thr at position 29 with a charged aminoacid; substitution of Thr at position 29 with a charged amino acidselected from the group consisting of Lys, Arg, His, Asp, Glu, cysteicacid, and homocysteic acid; substitution at position 29 with Asp, Glu,or Lys; substitution at position 29 with Glu; insertion of 1-3 chargedamino acids after position 29; insertion at position 30 (i.e., afterposition 29) of Glu or Lys; optionally with insertion at position 31 ofLys; addition of SEQ ID NO: 820 to the C-terminus, optionally, whereinthe amino acid at position 29 is Thr or Gly; substitution or addition ofan amino acid covalently attached to a hydrophilic moiety; or acombination thereof.

Any of the modifications described above in reference to Class 1glucagon agonists which increase glucagon receptor activity, retainpartial glucagon receptor activity, improve solubility, increasestability, or reduce degradation can be applied to Class 1 glucagonpeptides individually or in combination. Thus, Class 1 glucagon relatedpeptides can be prepared that retain at least 20% of the activity ofnative glucagon at the glucagon receptor, and which are soluble at aconcentration of at least 1 mg/mL at a pH between 6 and 8 or between 6and 9, (e.g. pH 7), and optionally retain at least 95% of the originalpeptide (e.g. 5% or less of the original peptide is degraded or cleaved)after 24 hours at 25° C. Alternatively, high potency Class 1 glucagonpeptides can be prepared that exhibit at least about 100%, 125%, 150%,175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%or 10-fold or more of the activity of native glucagon at the glucagonreceptor, and optionally are soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 or between 6 and 9, (e.g. pH 7), andoptionally retains at least 95% of the original peptide (e.g. 5% or lessof the original peptide is degraded or cleaved) after 24 hours at 25° C.In some embodiments, the Class 1 glucagon peptides described herein mayexhibit at least any of the above indicated relative levels of activityat the glucagon receptor but no more than 1,000%, 5,000% or 10,000% ofthe activity of native glucagon at the glucagon receptor.

Examples of Embodiments of Class 1 Glucagon Related Peptides

In accordance with some embodiments the native glucagon peptide of SEQID NO: 801 is modified by the substitution of the native amino acid atposition 28 and/or 29 with a negatively charged amino acid (e.g.,aspartic acid or glutamic acid) and optionally the addition of anegatively charged amino acid (e.g., aspartic acid or glutamic acid) tothe carboxy terminus of the peptide. In an alternative embodiment thenative glucagon peptide of SEQ ID NO: 801 is modified by thesubstitution of the native amino acid at position 29 with a positivelycharged amino acid (e.g., lysine, arginine or histidine) and optionallythe addition of one or two positively charged amino acid (e.g., lysine,arginine or histidine) on the carboxy terminus of the peptide. Inaccordance with some embodiments a glucagon analog having improvedsolubility and stability is provided wherein the analog comprises theamino acid sequence of SEQ ID NO: 834 with the proviso that at least oneamino acids at position, 28, or 29 is substituted with an acidic aminoacid and/or an additional acidic amino acid is added at the carboxyterminus of SEQ ID NO: 834. In some embodiments the acidic amino acidsare independently selected from the group consisting of Asp, Glu,cysteic acid and homocysteic acid.

In accordance with some embodiments a glucagon agonist having improvedsolubility and stability is provided wherein the agonist comprises theamino acid sequence of SEQ ID NO: 833, wherein at least one of the aminoacids at positions 27, 28 or 29 is substituted with a non-native aminoacid residue (i.e. at least one amino acid present at position 27, 28 or29 of the analog is an acid amino acid different from the amino acidpresent at the corresponding position in SEQ ID NO: 801). In accordancewith some embodiments a glucagon agonist is provided comprising thesequence of SEQ ID NO: 833 with the proviso that when the amino acid atposition 28 is asparagine and the amino acid at position 29 isthreonine, the peptide further comprises one to two amino acids,independently selected from the group consisting of Lys, Arg, His, Aspor Glu, added to the carboxy terminus of the glucagon peptide.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, applicants anticipate that one or moreof the amino acids located at positions at positions 2, 5, 7, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide ofSEQ ID NO: 811 can be substituted with an amino acid different from thatpresent in the native glucagon peptide, and still retain the enhancedpotency, physiological pH stability and biological activity of theparent glucagon peptide. For example, in accordance with someembodiments the methionine residue present at position 27 of the nativepeptide is changed to leucine or norleucine to prevent oxidativedegradation of the peptide.

In some embodiments a glucagon analog of SEQ ID NO: 833 is providedwherein 1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from thecorresponding amino acid of SEQ ID NO: 801. In accordance with anotherembodiment a glucagon analog of SEQ ID NO: 833 is provided wherein 1 to3 amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13, 14,16, 17, 18, 19, 20, 21 or 24 of the analog differ from the correspondingamino acid of SEQ ID NO: 801. In another embodiment, a glucagon analogof SEQ ID NO: 807, SEQ ID NO: 808 or SEQ ID NO: 834 is provided wherein1 to 2 amino acids selected from positions 1, 2, 5, 7, 10, 11, 12, 13,14, 16, 17, 18, 19, 20, 21 or 24 of the analog differ from thecorresponding amino acid of SEQ ID NO: 801, and in a further embodimentthose one to two differing amino acids represent conservative amino acidsubstitutions relative to the amino acid present in the native sequence(SEQ ID NO: 801). In some embodiments a glucagon peptide of SEQ ID NO:811 or SEQ ID NO: 813 is provided wherein the glucagon peptide furthercomprises one, two or three amino acid substitutions at positionsselected from positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20,21, 24, 27 or 29. In some embodiments the substitutions at positions 2,5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27 or 29 are conservativeamino acid substitutions.

In some embodiments a glucagon agonist is provided comprising an analogpeptide of SEQ ID NO: 801 wherein the analog differs from SEQ ID NO: 801by having an amino acid other than serine at position 2 and by having anacidic amino acid substituted for the native amino acid at position 28or 29 or an acidic amino acid added to the carboxy terminus of thepeptide of SEQ ID NO: 801. In some embodiments the acidic amino acid isaspartic acid or glutamic acid. In some embodiments a glucagon analog ofSEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832 isprovided wherein the analog differs from the parent molecule by asubstitution at position 2. More particularly, position 2 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of D-serine, alanine, D-alanine, glycine, n-methyl serine andamino isobutyric acid.

In another embodiment a glucagon agonist is provided comprising ananalog peptide of SEQ ID NO: 801 wherein the analog differs from SEQ IDNO: 801 by having an amino acid other than histidine at position 1 andby having an acidic amino acid substituted for the native amino acid atposition 28 or 29 or an acidic amino acid added to the carboxy terminusof the peptide of SEQ ID NO: 801. In some embodiments the acidic aminoacid is aspartic acid or glutamic acid. In some embodiments a glucagonanalog of SEQ ID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO:832 is provided wherein the analog differs from the parent molecule by asubstitution at position 1. More particularly, position 1 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of DMIA, D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine and homo-histidine.

In accordance with some embodiments the modified glucagon peptidecomprises a sequence selected from the group consisting of SEQ ID NO:809, SEQ ID NO: 812, SEQ ID NO: 813 and SEQ ID NO: 832. In a furtherembodiment a glucagon peptide is provided comprising a sequence of SEQID NO: 809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832 furthercomprising one to two amino acids, added to the C-terminus of SEQ ID NO:809, SEQ ID NO: 812, SEQ ID NO: 813 or SEQ ID NO: 832, wherein theadditional amino acids are independently selected from the groupconsisting of Lys, Arg, His, Asp Glu, cysteic acid or homocysteic acid.In some embodiments the additional amino acids added to the carboxyterminus are selected from the group consisting of Lys, Arg, His, Asp orGlu or in a further embodiment the additional amino acids are Asp orGlu.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 807 or a glucagon agonist analog thereof. In some embodiments thepeptide comprising a sequence selected from the group consisting of SEQID NO: 808, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812 and SEQ IDNO: 813. In another embodiment the peptide comprising a sequenceselected from the group consisting of SEQ ID NO: 808, SEQ ID NO: 810 andSEQ ID NO: 811. In some embodiments the glucagon peptide comprises thesequence of SEQ ID NO: 808, SEQ ID NO: 810 and SEQ ID NO: 811 furthercomprising an additional amino acid, selected from the group consistingof Asp and Glu, added to the C-terminus of the glucagon peptide. In someembodiments the glucagon peptide comprises the sequence of SEQ ID NO:811 or SEQ ID NO: 813, and in a further embodiment the glucagon peptidecomprises the sequence of SEQ ID NO: 811.

In accordance with some embodiments a glucagon agonist is providedcomprising a modified glucagon peptide selected from the groupconsisting of:

(SEQ ID NO: 834) NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R, (SEQ ID NO: 811)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R and (SEQ ID NO: 813)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Glu-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R

wherein Xaa at position 15 is Asp, Glu, cysteic acid, homoglutamic acidor homocysteic acid, the Xaa at position 28 is Asn or an acidic aminoacid and the Xaa at position 29 is Thr or an acidic amino acid and R isan acidic amino acid, COOH or CONH₂, with the proviso that an acidicacid residue is present at one of positions 28, 29 or 30. In someembodiments R is COOH, and in another embodiment R is CONH₂.

The present disclosure also encompasses glucagon fusion peptides whereina second peptide has been fused to the C-terminus of the glucagonpeptide to enhance the stability and solubility of the glucagon peptide.More particularly, the fusion glucagon peptide may comprise a glucagonagonist analog comprising a glucagon peptideNH₂—His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R(SEQ ID NO: 834), wherein R is an acidic amino acid or a bond and anamino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to the carboxy terminal aminoacid of the glucagon peptide. In some embodiments the glucagon peptideis selected from the group consisting of SEQ ID NO: 833, SEQ ID NO: 807or SEQ ID NO: 808 further comprising an amino acid sequence of SEQ IDNO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR)linked to the carboxy terminal amino acid of the glucagon peptide. Insome embodiments the glucagon fusion peptide comprises SEQ ID NO: 802,SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805 and SEQ ID NO: 806 or aglucagon agonist analog thereof, further comprising an amino acidsequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821 (KRNRNNIA) orSEQ ID NO: 822 (KRNR) linked to amino acid 29 of the glucagon peptide.In accordance with some embodiments the fusion peptide further comprisesa PEG chain linked to an amino acid at position 16, 17, 21, 24, 29,within a C-terminal extension, or at the C-terminal amino acid, whereinthe PEG chain is selected from the range of 500 to 40,000 Daltons. Insome embodiments the amino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS),SEQ ID NO: 821 (KRNRNNIA) or SEQ ID NO: 822 (KRNR) is bound to aminoacid 29 of the glucagon peptide through a peptide bond. In someembodiments the glucagon peptide portion of the glucagon fusion peptidecomprises a sequence selected from the group consisting of SEQ ID NO:810, SEQ ID NO: 811 and SEQ ID NO: 813. In some embodiments the glucagonpeptide portion of the glucagon fusion peptide comprises the sequence ofSEQ ID NO: 811 or SEQ ID NO: 813, wherein a PEG chain is linked atposition 21, 24, 29, within a C-terminal extension or at the C-terminalamino acid, respectively.

In another embodiment the glucagon peptide sequence of the fusionpeptide comprises the sequence of SEQ ID NO: 811, further comprising anamino acid sequence of SEQ ID NO: 820 (GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822 (KRNR) linked to amino acid 29 of theglucagon peptide. In some embodiments the glucagon fusion peptidecomprises a sequence selected from the group consisting of SEQ ID NO:824, SEQ ID NO: 825 and SEQ ID NO: 826. Typically the fusion peptides ofthe present invention will have a C-terminal amino acid with thestandard carboxylic acid group. However, analogs of those sequenceswherein the C-terminal amino acid has an amide substituted for thecarboxylic acid are also encompassed as embodiments. In accordance withsome embodiments the fusion glucagon peptide comprises a glucagonagonist analog selected from the group consisting of SEQ ID NO: 810, SEQID NO: 811 and SEQ ID NO: 813, further comprising an amino acid sequenceof SEQ ID NO: 823 (GPSSGAPPPS-CONH₂) linked to amino acid 29 of theglucagon peptide.

The glucagon agonists of the present invention can be further modifiedto improve the peptide's solubility and stability in aqueous solutionswhile retaining the biological activity of the glucagon peptide. Inaccordance with some embodiments, introduction of hydrophilic groups atone or more positions selected from positions 16, 17, 20, 21, 24 and 29of the peptide of SEQ ID NO: 811, or a glucagon agonist analog thereof,are anticipated to improve the solubility and stability of the pHstabilize glucagon analog. More particularly, in some embodiments theglucagon peptide of SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 813, orSEQ ID NO: 832 is modified to comprise one or more hydrophilic groupscovalently linked to the side chains of amino acids present at positions21 and 24 of the glucagon peptide.

In accordance with some embodiments, the glucagon peptide of SEQ ID NO:811 is modified to contain one or more amino acid substitution atpositions 16, 17, 20, 21, 24 and/or 29, wherein the native amino acid issubstituted with an amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG. Thenative peptide can be substituted with a naturally occurring amino acidor a synthetic (non-naturally occurring) amino acid. Synthetic ornon-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein.

In some embodiments, a glucagon agonist of SEQ ID NO: 810, SEQ ID NO:811 or SEQ ID NO: 813 is provided wherein the native glucagon peptidesequence has been modified to contain a naturally occurring or syntheticamino acid in at least one of positions 16, 17, 21, 24, 29, within aC-terminal extension or at the C-terminal amino acid of the nativesequence, wherein the amino acid substitute further comprises ahydrophilic moiety. In some embodiments the substitution is at position21 or 24, and in a further embodiment the hydrophilic moiety is a PEGchain. In some embodiments the glucagon peptide of SEQ ID NO: 811 issubstituted with at least one cysteine residue, wherein the side chainof the cysteine residue is further modified with a thiol reactivereagent, including for example, maleimido, vinyl sulfone, 2-pyridylthio,haloalkyl, and haloacyl. These thiol reactive reagents may containcarboxy, keto, hydroxyl, and ether groups as well as other hydrophilicmoieties such as polyethylene glycol units. In an alternativeembodiment, the native glucagon peptide is substituted with lysine, andthe side chain of the substituting lysine residue is further modifiedusing amine reactive reagents such as active esters (succinimido,anhydride, etc) of carboxylic acids or aldehydes of hydrophilic moietiessuch as polyethylene glycol. In some embodiments the glucagon peptide isselected form the group consisting of SEQ ID NO: 814, SEQ ID NO: 815,SEQ ID NO: 816, SEQ ID NO: 817, SEQ ID NO: 818 and SEQ ID NO: 819.

In accordance with some embodiments the pegylated glucagon peptidecomprises two or more polyethylene glycol chains covalently bound to theglucagon peptide wherein the total molecular weight of the glucagonchains is about 1,000 to about 5,000 Daltons. In some embodiments thepegylated glucagon agonist comprises a peptide of SEQ ID NO: 806,wherein a PEG chain is covalently linked to the amino acid residue atposition 21 and at position 24, and wherein the combined molecularweight of the two PEG chains is about 1,000 to about 5,000 Daltons. Inanother embodiment the pegylated glucagon agonist comprises a peptide ofSEQ ID NO: 806, wherein a PEG chain is covalently linked to the aminoacid residue at position 21 and at position 24, and wherein the combinedmolecular weight of the two PEG chains is about 5,000 to about 20,000Daltons.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with some embodiments the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 40,000 Daltons. In some embodiments the polyethyleneglycol chain has a molecular weight selected from the range of about 500to about 5,000 Daltons. In another embodiment the polyethylene glycolchain has a molecular weight of about 20,000 to about 40,000 Daltons.

Any of the glucagon peptides described above may be further modified toinclude a covalent or non-covalent intramolecular bridge or an alphahelix-stabilizing amino acid within the C-terminal portion of theglucagon peptide (amino acid positions 12-29). In accordance with someembodiments, the glucagon peptide comprises any one or more of themodifications discussed above in addition to an amino acid substitutionat positions 16, 20, 21, or 24 (or a combination thereof) with anα,α-disubstituted amino acid, e.g., Aib. In accordance with anotherembodiment, the glucagon peptide comprises any one or more modificationsdiscussed above in addition to an intramolecular bridge, e.g., a lactam,between the side chains of the amino acids at positions 16 and 20 of theglucagon peptide.

In accordance with some embodiments, the glucagon peptide comprises theamino acid sequence of SEQ ID NO: 877, wherein the Xaa at position 3 isan amino acid comprising a side chain of Structure I, II, or III:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃ alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, 0, or S; and Y isNHR⁴, SR³, or OR³. In some embodiments, X is NH or Y is NHR⁴. In someembodiments, R¹ is C₀₋₂ alkyl or C₁ heteroalkyl. In some embodiments, R²is NHR⁴ or C₁ alkyl. In some embodiments, R⁴ is H or C¹ alkyl. Inexemplary embodiments an amino acid comprising a side chain of StructureI is provided wherein, R¹ is CH₂—S, X is NH, and R² is CH₃(acetamidomethyl-cysteine, C(Acm)); R¹ is CH₂, X is NH, and R² is CH₃(acetyldiaminobutanoic acid, Dab(Ac)); R¹ is C₀ alkyl, X is NH, R² isNHR⁴, and R⁴ is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R¹ isCH₂—CH₂, X is NH, and R² is CH₃ (acetylornithine, Orn(Ac)). In exemplaryembodiments an amino acid comprising a side chain of Structure II isprovided, wherein R¹ is CH₂, Y is NHR⁴, and R⁴ is CH₃ (methylglutamine,Q(Me)); In exemplary embodiments an amino acid comprising a side chainof Structure III is provided wherein, R¹ is CH₂ and R⁴ is H(methionine-sulfoxide, M(O)); In specific embodiments, the amino acid atposition 3 is substituted with Dab(Ac). For example, glucagon agonistscan comprise the amino acid sequence of SEQ ID NO: 863, SEQ ID NO: 869,SEQ ID NO: 871, SEQ ID NO: 872, SEQ ID NO: 873, and SEQ ID NO: 874.

In certain embodiments, the glucagon peptide is an analog of theglucagon peptide of SEQ ID NO: 877. In specific aspects, the analogcomprises any of the amino acid modifications described herein,including, but not limited to: a substitution of Asn at position 28 witha charged amino acid; a substitution of Asn at position 28 with acharged amino acid selected from the group consisting of Lys, Arg, His,Asp, Glu, cysteic acid, and homocysteic acid; a substitution at position28 with Asn, Asp, or Glu; a substitution at position 28 with Asp; asubstitution at position 28 with Glu; a substitution of Thr at position29 with a charged amino acid; a substitution of Thr at position 29 witha charged amino acid selected from the group consisting of Lys, Arg,His, Asp, Glu, cysteic acid, and homocysteic acid; a substitution atposition 29 with Asp, Glu, or Lys; a substitution at position 29 withGlu; a insertion of 1-3 charged amino acids after position 29; aninsertion after position 29 of Glu or Lys; an insertion after position29 of Gly-Lys or Lys-Lys; and a combination thereof.

In certain embodiments, the analog of the glucagon peptide of SEQ ID NO:877 comprises an α,α-disubstituted amino acid, such as Aib, at one, two,three, or all of positions 16, 20, 21, and 24.

In certain embodiments, the analog of the glucagon peptide of SEQ ID NO:877 comprises one or more of the following: substitution of His atposition 1 with a non-native amino acid that reduces susceptibility ofthe glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),substitution of Ser at position 2 with a non-native amino acid thatreduces susceptibility of the glucagon peptide to cleavage by dipeptidylpeptidase IV (DPP-IV), substitution of Thr at position 7 with an aminoacid lacking a hydroxyl group, e.g., Abu or Ile; substitution of Tyr atposition 10 with Phe or Val; substitution of Lys at position 12 withArg; substitution of Asp at position 15 with Glu, substitution of Ser atposition 16 with Thr or Aib; substitution of Gln at position 20 with Alaor Aib; substitution of Asp at position 21 with Glu; substitution of Glnat position 24 with Ala or Aib; substitution of Met at position 27 withLeu or Nle; deletion of amino acids at positions 27-29; deletion ofamino acids at positions 28-29; deletion of the amino acid at positions29; addition of the amino acid sequence of SEQ ID NO: 820 to theC-terminus, wherein the amino acid at position 29 is Thr or Gly, or acombination thereof.

In accordance with specific embodiments, the glucagon peptide comprisesthe amino acid sequence of any of SEQ ID NOs: 862-867 and 869-874.

In certain embodiments, the analog of the glucagon peptide comprisingSEQ ID NO: 877 comprises a hydrophilic moiety, e.g., PEG, covalentlylinked to the amino acid at any of positions 16, 17, 20, 21, 24, and 29or at the C-terminal amino acid.

In certain embodiments, the analog of the glucagon peptide comprisingSEQ ID NO: 877 comprises an amino acid comprising a side chaincovalently attached, optionally, through a spacer, to an acyl group oran alkyl group, which acyl group or alkyl group is non-native to anaturally-occurring amino acid. The acyl group in some embodiments is aC4 to C30 fatty acyl group. In other embodiments, the alkyl group is aC4 to C30 alkyl. In specific aspects, the acyl group or alkyl group iscovalently attached to the side chain of the amino acid at position 10.In some embodiments, the amino acid at position 7 is Ile or Abu.

The glucagon agonist may be a peptide comprising the amino acid sequenceof any of the SEQ ID NOs: 801-919, optionally with up to 1, 2, 3, 4, or5 further modifications that retain glucagon agonist activity. Incertain embodiments, the glucagon agonist comprises the amino acids ofany of SEQ ID NOs: 859-919.

Class 2 Glucagon Related Peptides

In certain embodiments, the glucagon related peptide is a Class 2glucagon related peptide, which is described herein and in InternationalPatent Publication No. WO 2010/011439, and U.S. Application No.61/187,578, (filed on Jun. 16, 2009) the contents of which areincorporated by reference in their entirety.

The biological sequences referenced in the following section (SEQ IDNOs: 1001-1262) relating to Class 2 glucagon related peptides correspondto SEQ ID NOs: 1-262 in International Patent Publication No. WO2010/011439. SEQ ID NOs: 1263 to 1275 relating to Class 2 glucagonrelated peptides correspond to SEQ ID NOs: 657 to 669 in U.S.Application No. 61/187,578.

Activity

Native glucagon does not activate the GIP receptor, and normally hasabout 1% of the activity of native-GLP-1 at the GLP-1 receptor.Modifications to the native glucagon sequence described herein produceClass 2 glucagon related peptides that can exhibit potent glucagonactivity equivalent to or better than the activity of native glucagon(SEQ ID NO: 1001), potent GIP activity equivalent to or better than theactivity of native GIP (SEQ ID NO: 1004), and/or potent GLP-1 activityequivalent to or better than the activity of native GLP-1. In thisregard, the Class 2 glucagon related peptide may be one of aglucagon/GIP co-agonist, glucagon/GIP/GLP-1 tri-agonist, GIP/GLP-1co-agonist, or a GIP agonist glucagon peptide, as further describedherein.

In some embodiments, the Class 2 glucagon related peptides describedherein exhibit an EC₅₀ for GIP receptor activation activity of about 100nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less.In some embodiments, the Class 2 glucagon related peptides exhibit anEC₅₀ for glucagon receptor activation of about 100 nM or less, or about75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nM or less. In some embodiments,the Class 2 glucagon related peptides exhibit an EC₅₀ for GLP-1 receptoractivation of about 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4,3, 2 or 1 nM or less. Receptor activation can be measured by in vitroassays measuring cAMP induction in HEK293 cells over-expressing thereceptor, e.g. assaying HEK293 cells co-transfected with DNA encodingthe receptor and a luciferase gene linked to cAMP responsive element asdescribed in Example 2.

In some embodiments, Class 2 glucagon related peptides exhibit at leastabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175% or 200% or higheractivity at the GIP receptor relative to native GIP (GIP potency). Insome embodiments, the glucagon peptides described herein exhibit no morethan 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GIPreceptor relative to native GIP. In some embodiments, Class 2 glucagonrelated peptides exhibit at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or500% or higher activity at the glucagon receptor relative to nativeglucagon (glucagon potency). In some embodiments, the glucagon peptidesdescribed herein exhibit no more than 1000%, 10,000%, 100,000%, or1,000,000% activity at the glucagon receptor relative to nativeglucagon. In some embodiments, Class 2 glucagon related peptides exhibitat least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175% or 200% orhigher activity at the GLP-1 receptor relative to native GLP-1 (GLP-1potency). In some embodiments, the glucagon peptides described hereinexhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity atthe GLP-1 receptor relative to native GLP-1. A Class 2 glucagon relatedpeptide's activity at a receptor relative to a native ligand of thereceptor is calculated as the inverse ratio of EC₅₀s for the Class 2glucagon related peptide vs. the native ligand.

In some embodiments, Class 2 glucagon related peptides exhibit activityat both the glucagon receptor and the GIP receptor (“glucagon/GIPco-agonists”). These Class 2 glucagon related peptides have lost nativeglucagon's selectivity for glucagon receptor compared to GIP receptor.In some embodiments, the EC₅₀ of the Class 2 glucagon related peptide atthe GIP receptor is less than about 50-fold, 40-fold, 30-fold or 20-folddifferent (higher or lower) from its EC₅₀ at the glucagon receptor. Insome embodiments, the GIP potency of the Class 2 glucagon relatedpeptide is less than about 500-, 450-, 400-, 350-, 300-, 250-, 200-,150-, 100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher orlower) from its glucagon potency. In some embodiments, the ratio of theEC₅₀ of the Class 2 glucagon related peptide at the GIP receptor dividedby the EC₅₀ of the Class 2 glucagon related peptide at the glucagonreceptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5.In some embodiments, the ratio of the EC₅₀ at the GIP receptor dividedby the EC₅₀ at the glucagon receptor is about 1 or less than about 1(e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1,0.2). In some embodiments, the ratio of the GIP potency of the Class 2glucagon related peptide compared to the glucagon potency of the Class 2glucagon related peptide is less than about 500, 450, 400, 350, 300,250, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In someembodiments, the ratio of the potency at the GIP receptor divided by thepotency at the glucagon receptor is about 1 or less than about 1 (e.g.,about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). Insome embodiments, GLP-1 activity have been significantly reduced ordestroyed, e.g., by an amino acid modification at position 7, a deletionof the amino acid(s)C-terminal to the amino acid at position 27 or 28,yielding a 27- or 28-amino acid peptide, or a combination thereof.

In another aspect, Class 2 glucagon related peptides exhibit activity atthe glucagon, GIP and GLP-1 receptors (“glucagon/GIP/GLP-1tri-agonists”). These Class 2 glucagon related peptides have lost nativeglucagon's selectivity for the glucagon receptor compared to both theGLP-1 and GIP receptors. In some embodiments, the EC₅₀ of the Class 2glucagon related peptide at the GIP receptor is less than about 50-fold,40-fold, 30-fold or 20-fold different (higher or lower) from itsrespective EC₅₀s at the glucagon and GLP-1 receptors. In someembodiments, the GIP potency of the Class 2 glucagon related peptide isless than about 500-, 450-, 400-, 350-, 300-, 250-, 200-, 150-, 100-,75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher or lower) fromits glucagon and GLP-1 potencies. In some embodiments, the ratio of theEC₅₀ of the tri-agonist at the GIP receptor divided by the EC₅₀ of thetri-agonist at the GLP-1 receptor is less than about 100, 75, 60, 50,40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the EC₅₀ atthe GIP receptor divided by the EC₅₀ at the GLP-1 receptor is about 1 orless than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03,0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIPpotency of the tri-agonist compared to the GLP-1 potency of thetri-agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or5. In some embodiments, the ratio of the potency at the GIP receptordivided by the potency at the GLP-1 receptor is about 1 or less thanabout 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05,0.067, 0.1, 0.2). In related embodiments, the ratio of the EC₅₀ of thetri-agonist at the GIP receptor divided by the EC₅₀ of the tri-agonistat the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20,15, 10, or 5. In some embodiments, the ratio of the EC₅₀ at the GIPreceptor divided by the EC₅₀ at the glucagon receptor is about 1 or lessthan about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05,0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency ofthe tri-agonist compared to the glucagon potency of the tri-agonist isless than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50,40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the potencyat the GIP receptor divided by the potency at the glucagon receptor isabout 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02,0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio ofthe EC₅₀ of the tri-agonist at the GLP-1 receptor divided by the EC₅₀ ofthe tri-agonist at the glucagon receptor is less than about 100, 75, 60,50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the EC₅₀at the GLP-1 receptor divided by the EC₅₀ at the glucagon receptor isabout 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02,0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio ofthe GLP-1 potency of the tri-agonist compared to the glucagon potency ofthe tri-agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10,or 5. In some embodiments, the ratio of the potency at the GLP-1receptor divided by the potency at the glucagon receptor is about 1 orless than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03,0.05, 0.067, 0.1, 0.2).

In yet another aspect, Class 2 glucagon related peptides exhibitactivity at the GLP-1 and GIP receptors, but in which the glucagonactivity has been significantly reduced or destroyed (“GIP/GLP-1co-agonists”), e.g., by an amino acid modification at position 3. Forexample, substitution at this position with an acidic, basic, or ahydrophobic amino acid (glutamic acid, ornithine, norleucine) reducesglucagon activity. In some embodiments, the EC₅₀ of the glucagon peptideat the GIP receptor is less than about 50-fold, 40-fold, 30-fold or20-fold different (higher or lower) from its EC₅₀ at the GLP-1 receptor.In some embodiments, the GIP potency of the Class 2 glucagon relatedpeptide is less than about 25-, 20-, 15-, 10-, or 5-fold different(higher or lower) from its GLP-1 potency. In some embodiments theseClass 2 glucagon related peptides have about 10% or less of the activityof native glucagon at the glucagon receptor, e.g. about 1-10%, or about0.1-10%, or greater than about 0.1% but less than about 10%. In someembodiments, the ratio of the EC₅₀ of the Class 2 glucagon relatedpeptide at the GIP receptor divided by the EC₅₀ of the Class 2 glucagonrelated peptide at the GLP-1 receptor is less than about 100, 75, 60,50, 40, 30, 20, 15, 10, or 5, and no less than 1. In some embodiments,the ratio of the GIP potency of the Class 2 glucagon related peptidecompared to the GLP-1 potency of the Class 2 glucagon related peptide isless than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no lessthan 1.

In a further aspect, Class 2 glucagon related peptides exhibit activityat the GIP receptor, in which the glucagon and GLP-1 activity have beensignificantly reduced or destroyed (“GIP agonist glucagon peptides”),e.g., by amino acid modifications at positions 3 with Glu and 7 withIle. In some embodiments, these Class 2 glucagon related peptides haveabout 10% or less of the activity of native glucagon at the glucagonreceptor, e.g. about 1-10%, or about 0.1-10%, or greater than about0.1%, 0.5%, or 1% but less than about 1%, 5%, or 10%. In someembodiments these Class 2 glucagon related peptides also have about 10%or less of the activity of native GLP-1 at the GLP-1 receptor, e.g.about 1-10%, or about 0.1-10%, or greater than about 0.1%, 0.5%, or 1%but less than about 1%, 5%, or 10%.

In some embodiments, when the Class 2 glucagon related peptide is notpegylated, the EC₅₀ of the Class 2 glucagon related peptide for GIPreceptor activation is about 4, 2, 1 nM or less, or the analog has atleast about 1%, 2%, 3%, 4% or 5% of the activity of native GIP at theGIP receptor. In related embodiments, the EC₅₀ of the unpegylated Class2 glucagon related peptide for GLP-1 receptor activation is about 4, 2,1 nM or less or has at least about 1%, 2%, 3%, 4% or 5% of the activityof native GLP-1 at the GLP-1 receptor. In yet other related embodiments,the EC₅₀ of the unpegylated Class 2 glucagon related peptide forglucagon receptor activation is about 4, 2, 1 nM or less, or at leastabout 5%, 10%, 15% or 20% of the activity of native glucagon at theglucagon receptor. In some embodiments, the unpegylated Class 2 glucagonrelated peptide has less than about 1% of the activity of nativeglucagon at the glucagon receptor. In other embodiments, the unpegylatedClass 2 glucagon related peptide has less than about 10%, 5% or 1% ofthe activity of native GLP-1 at the GLP-1 receptor.

In embodiments where the Class 2 glucagon related peptides are linked tohydrophilic moieties such as PEG, the relative EC₅₀s at one or morereceptors may be higher e.g., about 10-fold higher. For example, theEC₅₀ of a pegylated analog for GIP receptor activation is about 10 nM orless, or the Class 2 glucagon related peptide has at least about 0.1%,0.2%, 0.3%, 0.4% or 0.5% of the activity of native GIP at the GIPreceptor. In related embodiments, the EC₅₀ of a pegylated Class 2glucagon related peptide for GLP-1 receptor activation is about 10 nM orless or has at least about 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of theactivity of native GLP-1 at the GLP-1 receptor. In yet other relatedembodiments, the EC₅₀ of a pegylated Class 2 glucagon related peptidefor glucagon receptor activation is about 10 nM or less, or at leastabout 0.5%, 1%, 1.5% or 2% of the activity of native glucagon at theglucagon receptor. In some embodiments, the Class 2 glucagon relatedpeptide has less than about 1% of the activity of native glucagon at theglucagon receptor. In other embodiments, the Class 2 glucagon relatedpeptide has less than about 10%, 5% or 1% of the activity of nativeGLP-1 at the GLP-1 receptor.

Modifications

The modifications disclosed herein in reference to a Class 2 glucagonrelated peptide permit the manipulation of glucagon (SEQ ID NO: 1001) tocreate glucagon peptides that exhibit increased GIP activity, glucagonactivity, and/or GLP-1 activity. Other modifications disclosed herein inreference to a Class 2 glucagon related peptide prolong the half-life,increase solubility, or increase stability of the resulting peptide. Yetother modifications disclosed herein in reference to a Class 2 glucagonrelated peptide have no effect on activity, or can be made withoutdestroying the desired activity or activities. Any of the combinationsin reference to a Class 2 glucagon related peptide that serve the samepurpose (e.g. increasing GIP activity) can be applied individually or incombination. Any of the single or sets of combinations in reference to aClass 2 glucagon related peptide that confer enhanced properties can beapplied individually or in combination, e.g. increased GIP and/or GLP-1activity can be combined with increased half-life. In relatedembodiments, 1, 2, 3, 4, 5, 6 or more of the amino acid modificationsmay be non-conservative substitutions, additions or deletions. In someembodiments, 1, 2, 3, 4, 5, 6 or more of the amino acid modificationsmay be conservative substitutions.

Modifications that Affect GIP Activity

Enhanced activity at the GIP receptor is provided by an amino acidmodification at position 1. For example, His at position 1 issubstituted with a large, aromatic amino acid, optionally Tyr, Phe, Trp,amino-Phe, nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr,or 3-amino Tyr. The combination of Tyr at position 1 with stabilizationof the alpha helix within the region corresponding to amino acids 12-29provided a Class 2 glucagon related peptide that activates the GIPreceptor as well as the GLP-1 receptor and the glucagon receptor. Thealpha helix structure can be stabilized by, e.g., formation of acovalent or non-covalent intramolecular bridge, or substitution and/orinsertion of amino acids around positions 12-29 with an alphahelix-stabilizing amino acid (e.g., an α,α-disubstituted amino acid).

Enhanced activity at the GIP receptor is also provided by amino acidmodifications at positions 27 and/or 28, and optionally at position 29.For example, the Met at position 27 is substituted with a largealiphatic amino acid, optionally Leu, the Asn at position 28 issubstituted with a small aliphatic amino acid, optionally Ala, and theThr at position 29 is substituted with a small aliphatic amino acid,optionally Gly. Substitution with LAG at positions 27-29 providesincreased GIP activity relative to the native MNT sequence at thosepositions.

Enhanced activity at the GIP receptor is also provided by an amino acidmodification at position 12. For example, position 12 is substitutedwith a large, aliphatic, nonpolar amino acid, optionally Ile.

Enhanced activity at the GIP receptor is also provided by an amino acidmodification at positions 17 and/or 18. For example, position 17 issubstituted with a polar residue, optionally Gln, and position 18 issubstituted with a small aliphatic amino acid, optionally Ala. Asubstitution with QA at positions 17 and 18 provides increased GIPactivity relative to the native RR sequence at those positions.

Increased activity at the GIP receptor is provided by modifications thatpermit formation of an intramolecular bridge between amino acid sidechains at positions from 12 to 29. For example, an intramolecular bridgecan be formed by a covalent bond between the side chains of two aminoacids at positions i and i+4 or between positions j and j+3, or betweenpositions k and k+7. In exemplary embodiments, the bridge is betweenpositions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20. Inother embodiments, non-covalent interactions such as salt bridges can beformed between positively and negatively charged amino acids at thesepositions.

Any of the modifications described above which increase GIP receptoractivity can be applied individually or in combination. Combinations ofthe modifications that increase GIP receptor activity generally providehigher GIP activity than any of such modifications taken alone.

Modifications that Affect Glucagon Activity

In some embodiments, enhanced glucagon potency is provided by an aminoacid modification at position 16 of native glucagon (SEQ ID NO: 1001).By way of nonlimiting example, such enhanced potency can be provided bysubstituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms. In some embodimentsthe glucagon peptide retains its original selectivity for the glucagonreceptor relative to the GLP-1 receptors.

Glucagon receptor activity can be reduced by an amino acid modificationat position 3, e.g. substitution of the naturally occurring glutamine atposition 3, with an acidic, basic, or a hydrophobic amino acid. Forexample substitution at position 3 with glutamic acid, ornithine, ornorleucine substantially reduces or destroys glucagon receptor activity.

Maintained or enhanced activity at the glucagon receptor may be achievedby modifying the Gln at position 3 with a glutamine analog, as describedherein. For example, glucagon agonists can comprise the amino acidsequence of any of SEQ ID NOs: 1243-1248, 1250, 1251, and 1253-1256.

Restoration of glucagon activity which has been reduced by amino acidmodifications at positions 1 and 2 is provided by modifications thatthat stabilize the alpha helix structure of the C-terminal portion(amino acids 12-29) of the glucagon peptide or analog thereof. Forexample, an intramolecular bridge can be formed by a covalent bondbetween the side chains of two amino acids at positions i and i+4 orbetween positions j and j+3, or between positions k and k+7. In otherembodiments, non-covalent interactions such as salt bridges can beformed between positively and negatively charged amino acids at thesepositions. In yet other embodiments, one or more α,α-disubstituted aminoacids are inserted or substituted into this C-terminal portion (aminoacids 12-29) at positions that retain the desired activity. For example,one, two, three or all of positions 16, 20, 21 or 24 are substitutedwith an α,α-disubstituted amino acid, e.g., Aib.

Modifications that Affect GLP-1 Activity

Enhanced activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester.

Enhanced activity at the GLP-1 receptor is also provided by stabilizingthe alpha-helix structure in the C-terminal portion of glucagon (aroundamino acids 12-29), e.g., through formation of an intramolecular bridgebetween the side chains of two amino acids, or substitution and/orinsertion of amino acids around positions 12-29 with an alphahelix-stabilizing amino acid (e.g., an α,α-disubstituted amino acid), asfurther described herein. In exemplary embodiments, the side chains ofthe amino acid pairs 12 and 16, 13 and 17, 16 and 20, 17 and 21, 20 and24 or 24 and 28 (amino acid pairs in which i=12, 16, 20, or 24) arelinked to one another and thus stabilize the glucagon alpha helix. Insome embodiments, the bridge or linker is about 8 (or about 7-9) atomsin length, particularly when the bridge is between positions i and i+4.In some embodiments, the bridge or linker is about 6 (or about 5-7)atoms in length, particularly when the bridge is between positions j andj+3.

In some embodiments, intramolecular bridges are formed by (a)substituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms, and (b) substitutingthe naturally occurring glutamine at position 20 with anotherhydrophilic amino acid having a side chain that is either charged or hasan ability to hydrogen-bond, and is at least about 5 (or about 4-6)atoms in length, for example, lysine, citrulline, arginine, orornithine. The side chains of such amino acids at positions 16 and 20can form a salt bridge or can be covalently linked. In some embodimentsthe two amino acids are bound to one another to form a lactam ring.

In some embodiments, stabilization of the alpha helix structure in theC-terminal portion of the glucagon peptide is achieved through theformation of an intramolecular bridge other than a lactam bridge. Forexample, suitable covalent bonding methods include any one or more ofolefin metathesis, lanthionine-based cyclization, disulfide bridge ormodified sulfur-containing bridge formation, the use of a,co-diaminoalkane tethers, the formation of metal-atom bridges, and othermeans of peptide cyclization are used to stabilize the alpha helix.

In yet other embodiments, one or more α,α-disubstituted amino acids areinserted or substituted into this C-terminal portion (amino acids 12-29)at positions that retain the desired activity. For example, one, two,three or all of positions 16, 20, 21 or 24 are substituted with anα,α-disubstituted amino acid, e.g., Aib.

Increased activity at the GLP-1 receptor is provided by an amino acidmodification at position 20 as described herein.

Increased activity at the GLP-1 receptor is provided by addingGPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096) to theC-terminus. GLP-1 activity in such analogs can be further increased bymodifying the amino acid at position 18, 28 or 29, or at position 18 and29, as described herein.

A further modest increase in GLP-1 potency is provided by modifying theamino acid at position 10 to be a large, aromatic amino acid residue,optionally Trp.

Reduced activity at the GLP-1 receptor is provided, e.g., by an aminoacid modification at position 7 as described herein.

Potency at the GLP-1 receptor can be further enhanced by an alaninesubstitution for the native arginine at position 18.

Any of the modifications described above in reference to a Class 2glucagon related peptide which increase GLP-1 receptor activity can beapplied individually or in combination. Combinations of themodifications that increase GLP-1 receptor activity generally providehigher GLP-1 activity than any of such modifications taken alone. Forexample, the invention provides glucagon peptides that comprisemodifications at position 16, at position 20, and at the C-terminalcarboxylic acid group, optionally with a covalent bond between the aminoacids at positions 16 and 20; glucagon peptides that comprisemodifications at position 16 and at the C-terminal carboxylic acidgroup; glucagon peptides that comprise modifications at positions 16 and20, optionally with a covalent bond between the amino acids at positions16 and 20; and glucagon peptides that comprise modifications at position20 and at the C-terminal carboxylic acid group.

Modifications that Improve DPP-IV Resistance

Modifications at position 1 and/or 2 can increase the peptide'sresistance to dipeptidyl peptidase IV (DPP IV) cleavage. For example,position 1 and/or position 2 may be substituted with a DPP-IV resistantamino acid as described herein. In some embodiments, the amino acid atposition 2 is substituted with N-methyl alanine.

It was observed that modifications at position 2 (e.g. Aib at position2) and in some cases modifications at position 1 (e.g., DMIA atposition 1) may reduce glucagon activity, sometimes significantly;surprisingly, this reduction in glucagon activity can be restored bystabilizing the alpha-helix structure in the C-terminal portion ofglucagon (around amino acids 12-29), e.g., through formation of acovalent bond between the side chains of two amino acids, as describedherein. In some embodiments, the covalent bond is between amino acids atpositions “i” and “i+4”, or positions “j” and “j+3”, e.g., betweenpositions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20. Inexemplary embodiments, this covalent bond is a lactam bridge between aglutamic acid at position 16 and a lysine at position 20. In someembodiments, this covalent bond is an intramolecular bridge other than alactam bridge, as described herein.

Modifications that Reduce Degradation

In yet further exemplary embodiments, any of the Class 2 glucagonrelated peptides can be further modified to improve stability bymodifying the amino acid at position 15 and/or 16 of SEQ ID NO: 1001 toreduce degradation of the peptide over time, especially in acidic oralkaline buffers. Such modifications reduce cleavage of the Asp15-Ser16peptide bond. In exemplary embodiments, the amino acid modification atposition 15 is a deletion or substitution of Asp with glutamic acid,homoglutamic acid, cysteic acid or homocysteic acid. In other exemplaryembodiments, the amino acid modification at position 16 is a deletion orsubstitution of Ser with Thr or Aib. In other exemplary embodiments, Serat position 16 is substituted with glutamic acid or with anothernegatively charged amino acid having a side chain with a length of 4atoms, or alternatively with any one of glutamine, homoglutamic acid, orhomocysteic acid.

In some embodiments, the methionine residue present at position 27 ofthe native peptide is modified, e.g. by deletion or substitution. Suchmodifications may prevent oxidative degradation of the peptide. In someembodiments, the Met at position 27 is substituted with leucine,isoleucine or norleucine. In some specific embodiments, Met at position27 is substituted with leucine or norleucine.

In some embodiments, the Gln at position 20 and/or 24 is modified, e.g.by deletion or substitution. Such modifications can reduce degradationthat occurs through deamidation of Gln. In some embodiments, the Gln atposition 20 and/or 24 is substituted with Ser, Thr, Ala or Aib. In someembodiments the Gln at position 20 and/or 24 is substituted with Lys,Arg, Orn, or Citrulline.

In some embodiments, the Asp at position 21 is modified, e.g. bydeletion or substitution. Such modifications can reduce degradation thatoccurs through dehydration of Asp to form a cyclic succinimideintermediate followed by isomerization to iso-aspartate. In someembodiments, position 21 is substituted with Glu, homoglutamic acid orhomocysteic acid. In some specific embodiments, position 21 issubstituted with Glu.

Stabilization of the Alpha Helix Structure

Stabilization of the alpha-helix structure in the C-terminal portion ofthe Class 2 glucagon related peptide (around amino acids 12-29) providesenhanced GLP-1 and/or GIP activity and restores glucagon activity whichhas been reduced by amino acid modifications at positions 1 and/or 2.The alpha helix structure can be stabilized by, e.g., formation of acovalent or non-covalent intramolecular bridge, or substitution and/orinsertion of amino acids around positions 12-29 with an alphahelix-stabilizing amino acid (e.g., an α,α-disubstituted amino acid).Stabilization of the alpha-helix structure of a GIP agonist may beaccomplished as described herein.

Acylation and Alkylation

In accordance with some embodiments, the glucagon peptides disclosedherein are modified to comprise an acyl group or alkyl group, e.g., anacyl or alkyl group which is non-native to a naturally-occurring aminoacid as described herein. Acylation or alkylation can increase thehalf-life of the glucagon peptides in circulation. Acylation oralkylation can advantageously delay the onset of action and/or extendthe duration of action at the glucagon and/or GLP-1 receptors and/orimprove resistance to proteases such as DPP-IV and/or improvesolubility. Activity at the glucagon and/or GLP-1 and/or GIP receptorsof the glucagon peptide may be maintained after acylation. In someembodiments, the potency of the acylated glucagon peptides is comparableto the unacylated versions of the glucagon peptides. Class 2 glucagonrelated peptides may be acylated or alkylated at the same amino acidposition where a hydrophilic moiety is linked, or at a different aminoacid position, as described herein.

In some embodiments, the invention provides a glucagon peptide modifiedto comprise an acyl group or alkyl group covalently linked to the aminoacid at position 10 of the glucagon peptide. The glucagon peptide mayfurther comprise a spacer between the amino acid at position 10 of theglucagon peptide and the acyl group or alkyl group. In some embodiments,the acyl group is a fatty acid or bile acid, or salt thereof, e.g. a C4to C30 fatty acid, a C8 to C24 fatty acid, cholic acid, a C4 to C30alkyl, a C8 to C24 alkyl, or an alkyl comprising a steroid moiety of abile acid. The spacer is any moiety with suitable reactive groups forattaching acyl or alkyl groups. In exemplary embodiments, the spacercomprises an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional, or a hydrophobic bifunctional spacer. In some embodiments,the spacer is selected from the group consisting of: Trp, Glu, Asp, Cysand a spacer comprising NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is anyinteger from 1 to 6 and n is any integer from 2 to 12. Such acylated oralkylated glucagon peptides may also further comprise a hydrophilicmoiety, optionally a polyethylene glycol. Any of the foregoing glucagonpeptides may comprise two acyl groups or two alkyl groups, or acombination thereof.

Conjugates and Fusions

The GIP agonist can be linked, optionally via covalent bonding andoptionally via a linker, to a conjugate moiety as described herein.

In other embodiments, the second peptide is XGPSSGAPPPS (SEQ ID NO:1096), wherein X is selected from one of the 20 common amino acids,e.g., glutamic acid, aspartic acid or glycine. In some embodiments Xrepresents an amino acid, for example Cys, that further comprises ahydrophilic moiety covalently linked to the side chain of that aminoacid. Such C-terminal extensions improve solubility and also can improveGIP or GLP-1 activity. In some embodiments wherein the glucagon peptidefurther comprises a carboxy terminal extension, the carboxy terminalamino acid of the extension ends in an amide group or an ester grouprather than a carboxylic acid.

In some embodiments, e.g., in glucagon peptides which comprise theC-terminal extension, the threonine at position 29 of the nativeglucagon peptide is replaced with a glycine. For example, a glucagonpeptide having a glycine substitution for threonine at position 29 andcomprising the C-terminal extension of GPSSGAPPPS (SEQ ID NO: 1095) isfour times as potent at the GLP-1 receptor as native glucagon modifiedto comprise the same C-terminal extension. This T29G substitution can beused in conjunction with other modifications disclosed herein to enhancethe affinity of the glucagon peptides for the GLP-1 receptor. Forexample, the T29G substitution can be combined with the S16E and N20Kamino acid substitutions, optionally with a lactam bridge between aminoacids 16 and 20, and optionally with addition of a PEG chain asdescribed herein.

In some embodiments an amino acid is added to the C-terminus, and theadditional amino acid is selected from the group consisting of glutamicacid, aspartic acid and glycine.

Modifications that Enhance Solubility

In another embodiment, the solubility of any of the glucagon peptidescan be improved by amino acid substitutions and/or additions thatintroduce a charged amino acid into the C-terminal portion of thepeptide, preferably at a position C-terminal to position 27 of SEQ IDNO: 1001. Optionally, one, two or three charged amino acids may beintroduced within the C-terminal portion, preferably C-terminal toposition 27. In some embodiments the native amino acid(s) at positions28 and/or 29 are substituted with one or two charged amino acids, and/orin a further embodiment one to three charged amino acids are also addedto the C-terminus of the peptide. In exemplary embodiments, one, two orall of the charged amino acids are negatively charged. In someembodiments, the negatively charged (acidic amino acid) is aspartic acidor glutamic acid.

Additional modifications, e.g. conservative substitutions, may be madeto the glucagon peptide that still allow it to retain GIP activity (andoptionally GLP-1 activity and/or glucagon activity).

Other Modifications

Any of the modifications described above in reference to a Class 2peptide which increase or decrease GIP activity, which increase ordecrease glucagon receptor activity, and which increase GLP-1 receptoractivity can be applied individually or in combination. Any of themodifications described above in reference to a Class 2 glucagon relatedpeptide can also be combined with other modifications that confer otherdesirable properties, such as increased solubility and/or stabilityand/or duration of action, as described herein with regard to Class 2glucagon related peptides. Alternatively, any of the modificationsdescribed above in reference to Class 2 glucaton related peptides can becombined with other modifications described herein in reference to Class2 glucagon related peptides that do not substantially affect solubilityor stability or activity. Exemplary modifications include but are notlimited to:

(A) Improving solubility, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. Such a charged aminoacid can be introduced by substituting a native amino acid with acharged amino acid, e.g. at positions 28 or 29, or alternatively byadding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged. Such modificationsincrease solubility, e.g. provide at least 2-fold, 5-fold, 10-fold,15-fold, 25-fold, 30-fold or greater solubility relative to nativeglucagon at a given pH between about 5.5 and 8, e.g., pH 7, whenmeasured after 24 hours at 25° C.

(B) Increasing solubility and duration of action or half-life incirculation by addition of a hydrophilic moiety such as a polyethyleneglycol chain, as described herein, e.g. at position 16, 17, 20, 21, 24or 29, within a C-terminal extension, or at the C-terminal amino acid ofthe peptide,

(C) Increasing solubility and/or duration of action or half-life incirculation and/or delaying the onset of action by acylation oralkylation of the glucagon peptide, as described herein;

(D) Increasing duration of action or half-life in circulation throughintroducing resistance to dipeptidyl peptidase IV (DPP IV) cleavage bymodification of the amino acid at position 1 or 2 as described herein.

(E) Increasing stability by modification of the Asp at position 15, forexample, by deletion or substitution with glutamic acid, homoglutamicacid, cysteic acid or homocysteic acid. Such modifications can reducedegradation or cleavage at a pH within the range of 5.5 to 8, forexample, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, upto 100% of the original peptide after 24 hours at 25° C. Suchmodifications reduce cleavage of the peptide bond between Asp15-Ser16.

(F) Increasing stability by modification of the Ser at position 16, forexample by substitution with Thr or Aib. Such modifications also reducecleavage of the peptide bond between Asp15-Ser16.

(G) Increasing stability by modification of the methionine at position27, for example, by substitution with leucine or norleucine. Suchmodifications can reduce oxidative degradation. Stability can also beincreased by modification of the Gln at position 20 or 24, e.g. bysubstitution with Ser, Thr, Ala or Aib. Such modifications can reducedegradation that occurs through deamidation of Gln. Stability can beincreased by modification of Asp at position 21, e.g. by substitutionwith Glu. Such modifications can reduce degradation that occurs throughdehydration of Asp to form a cyclic succinimide intermediate followed byisomerization to iso-aspartate.

(H) Non-conservative or conservative substitutions, additions ordeletions that do not substantially affect activity, for example,conservative substitutions at one or more of positions 2, 5, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; substitutionof one or more of these positions with Ala; deletion of amino acids atone or more of positions 27, 28 or 29; or deletion of amino acid 29optionally combined with a C-terminal amide or ester in place of theC-terminal carboxylic acid group; substitution of Lys at position 12with Arg; substitution of Tyr at position 10 with Val or Phe;

Preservation of activity after pegylation is provided by the addition ofGPSSGAPPPS (SEQ ID NO: 1095) to the C-terminus.

Some positions of the native glucagon peptide can be modified whileretaining at least some of the activities of the parent peptide.Accordingly, applicants anticipate that one or more of the amino acidslocated at positions at positions 2, 5, 10, 11, 12, 13, 14, 17, 18, 19,20, 21, 24, 27, 28 or 29 can be substituted with an amino acid differentfrom that present in the native glucagon peptide, and still retainactivity at the glucagon receptor.

In some embodiments, position 18 is substituted with an amino acidselected from the group consisting of Ala, Ser, or Thr. In someembodiments the amino acid at position 20 is substituted with Ser, Thr,Lys, Arg, Orn, Citrulline or Aib. In some embodiments, position 21 issubstituted with Glu, homoglutamic acid or homocysteic acid. In someembodiments, the glucagon peptide comprises 1 to 10 amino acidmodifications selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28and 29. In exemplary embodiments, the modifications are one or moreamino acid substitutions selected from the group consisting of Gln17,Ala18, Glu21, Ile23, Ala24, Val27 and Gly29. In some embodiments, 1 to 2amino acids selected from positions 17-26 differ from the parentpeptide. In other embodiments, 1 to 2 amino acids selected frompositions 17-22 differ from the parent peptide. In yet otherembodiments, the modifications are Gln17, Ala18, Glu21, Ile23 and Ala24.

In some embodiments, one or more amino acids is added to the carboxyterminus of the glucagon peptide. The amino acid is typically selectedfrom one of the 20 common amino acids, and in some embodiments the aminoacid has an amide group in place of the carboxylic acid of the nativeamino acid. In exemplary embodiments the added amino acid is selectedfrom the group consisting of glutamic acid and aspartic acid andglycine.

Other Modifications that do not Destroy Activity Include W10 or R20.

In some embodiments, the Class 2 glucagon related peptides disclosedherein are modified by truncation of the C-terminus by one or two aminoacid residues yet retain similar activity and potency at the glucagon,GLP-1 and/or GIP receptors. In this regard, the amino acid at position29 and/or 28 can be deleted.

Exemplary Embodiments

In accordance with some embodiments of the invention, the analog ofglucagon (SEQ ID NO: 1001) having GIP agonist activity comprises SEQ IDNO: 1001 with (a) an amino acid modification at position 1 that confersGIP agonist activity, (b) a modification which stabilizes the alphahelix structure of the C-terminal portion (amino acids 12-29) of theanalog, and (c) optionally, 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10) further amino acid modifications. In some embodiments, the analogexhibits at least about 1% activity of native GIP at the GIP receptor orany other activity level at the GIP receptor described herein.

In certain embodiments, the modification which stabilizes the alphahelix structure is one which provides or introduces an intramolecularbridge, including, for example, a covalent intramolecular bridge, suchas any of those described herein. The covalent intramolecular bridge insome embodiments is a lactam bridge. The lactam bridge of the analog ofthese embodiments can be a lactam bridge as described herein. See, e.g.,the teachings of lactam bridges under the section “Stabilization of theAlpha Helix Structure.” For example, the lactam bridge may be one whichis between the side chains of amino acids at positions i and i+4 orbetween the side chains of amino acids at positions j and j+3, wherein iis 12, 13, 16, 17, 20 or 24, and wherein j is 17. In certainembodiments, the lactam bridge can be between the amino acids atpositions 16 and 20, wherein one of the amino acids at positions 16 and20 is substituted with Glu and the other of the amino acids at positions16 and 20 is substituted with Lys.

In alternative embodiments, the modification which stabilizes the alphahelix structure is the introduction of one, two, three, or fourα,α-disubstituted amino acids at position(s) 16, 20, 21, and 24 of theanalog. In some embodiments, the α,α-disubstituted amino acid is Aib. Incertain aspects, the α,α-disubstituted amino acid (e.g., Aib) is atposition 20 and the amino acid at position 16 is substituted with apositive-charged amino acid, such as, for example, an amino acid ofFormula IV, which is described herein. The amino acid of Formula IV maybe homoLys, Lys, Orn, or 2,4-diaminobutyric acid (Dab).

In specific aspects of the invention, the amino acid modification atposition 1 is a substitution of His with an amino acid lacking animidazole side chain, e.g. a large, aromatic amino acid (e.g., Tyr).

In certain aspects, the analog of glucagon comprises amino acidmodifications at one, two or all of positions 27, 28 and 29. Forexample, the Met at position 27 can be substituted with a largealiphatic amino acid, optionally Leu, the Asn at position 28 can besubstituted with a small aliphatic amino acid, optionally Ala, the Thrat position 29 can be substituted with a small aliphatic amino acid,optionally Gly, or a combination of two or three of the foregoing. Inspecific embodiments, the analog of glucagon comprises Leu at position27, Ala at position 28, and Gly or Thr at position 29.

In certain embodiments of the invention, the analog of glucagoncomprises an extension of 1 to 21 amino acids C-terminal to the aminoacid at position 29. The extension can comprise the amino acid sequenceof SEQ ID NO: 1095 or 1096, for instance. Additionally or alternatively,the analog of glucagon can comprise an extension of which 1-6 aminoacids of the extension are positive-charged amino acids. Thepositive-charged amino acids may be amino acids of Formula IV,including, but not limited to Lys, homoLys, Orn, and Dab.

The analog of glucagon in some embodiments is acylated or alkylated asdescribed herein. For instance, the acyl or alkyl group may be attachedto the analog of glucagon, with or without a spacer, at position 10 or40 of the analog, as further described herein. The analog mayadditionally or alternatively be modified to comprise a hydrophilicmoiety as further described herein. Furthermore, in some embodiments,the analog comprises any one or a combination of the followingmodifications:

(a) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,N-methyl-Ser, Aib, Val, or α-amino-N-butyric acid;

(b) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe, or Val:

(c) Linkage of an acyl group to a Lys at position 10;

(d) Lys at position 12 substituted with Arg or Ile;

(e) Ser at position 16 substituted with Glu, Gln, homoglutamic acid,homocysteic acid, Thr, Gly, or Aib;

(f) Arg at position 17 substituted with Gln;

(g) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;

(h) Gln at position 20 substituted with Ser, Thr, Ala, Lys, Citrulline,Arg, Orn, or Aib;

(i) Asp at position 21 substituted with Glu, homoglutamic acid,homocysteic acid;

(j) Val at position 23 substituted with Ile;

(k) Gln at position 24 substituted with Asn, Ser, Thr, Ala, or Aib;

(l) and a conservative substitution at any of positions 2 5, 9, 10, 11,12. 13, 14, 15, 16, 8 19 20, 21. 24, 27, 28, and 29.

In exemplary embodiments, the analog of glucagon (SEQ ID NO: 1001)having GIP agonist activity comprises the following modifications:

(a) an amino acid modification at position 1 that confers GIP agonistactivity,

(b) a lactam bridge between the side chains of amino acids at positionsi and i+4 or between the side chains of amino acids at positions j andj+3, wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17,

(c) amino acid modifications at one, two or all of positions 27, 28 and29, e.g., amino acid modifications at position 27 and/or 28, and

(d) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3, 4, 5, 6,7, 8 or 9 further amino acid modifications,

and the EC₅₀ of the analog for GIP receptor activation is about 10 nM orless.

The lactam bridge of the analog of these embodiments can be a lactambridge as described herein. See, e.g., the teachings of lactam bridgesunder the section “Stabilization of the Alpha Helix Structure.” Forexample, the lactam bridge can be between the amino acids at positions16 and 20, wherein one of the amino acids at positions 16 and 20 issubstituted with Glu and the other of the amino acids at positions 16and 20 is substituted with Lys.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 1005-1094.

In other exemplary embodiments, the analog of glucagon (SEQ ID NO: 1001)having GIP agonist activity comprises the following modifications:

(a) an amino acid modification at position 1 that confers GIP agonistactivity,

(b) one, two, three, or all of the amino acids at positions 16, 20, 21,and 24 of the analog is substituted with an α,α-disubstituted aminoacid,

(c) amino acid modifications at one, two or all of positions 27, 28 and29, e.g., amino acid modifications at position 27 and/or 28, and

(d) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3, 4, 5, 6,7, 8 or 9 further amino acid modifications,

and the EC₅₀ of the analog for GIP receptor activation is about 10 nM orless.

The α,α-disubstituted amino acid of the analog of these embodiments canbe any α,α-disubstituted amino acid, including, but not limited to,amino iso-butyric acid (Aib), an amino acid disubstituted with the sameor a different group selected from methyl, ethyl, propyl, and n-butyl,or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In certain embodiments, theα,α-disubstituted amino acid is Aib. In certain embodiments, the aminoacid at position 20 is substituted with an α,α-disubstituted amino acid,e.g., Aib.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 1099-1141,1144-1164, 1166-1169, and 1173-1178.

In yet other exemplary embodiments, the analog of glucagon (SEQ ID NO:1001) having GIP agonist activity comprises the following modifications:

(a) an amino acid modification at position 1 that confers GIP agonistactivity,

(b) an amino acid substitution of Ser at position 16 with an amino acidof Formula IV:

wherein n is 1 to 16, or 1 to 10, or 1 to 7, or 1 to 6, or 2 to 6, eachof R1 and R2 is independently selected from the group consisting of H,C1-C18 alkyl, (C1-C18 alkyl)OH, (C1-C18 alkyl)NH2, (C1-C18 alkyl)SH,(C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic),(C0-C4 alkyl)(C6-C10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl),wherein R7 is H or OH, and the side chain of the amino acid of FormulaIV comprises a free amino group,

(c) an amino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid,

(d) amino acid modifications at one, two or all of positions 27, 28 and29, e.g., amino acid modifications at position 27 and/or 28, and

(e) 1-9 or 1-6 further amino acid modifications, e.g. 1, 2, 3, 4, 5, 6,7, 8 or 9 further amino acid modifications,

and the EC₅₀ of the analog for GIP receptor activation is about 10 nM orless.

The amino acid of Formula IV of the analog of these embodiments may beany amino acid, such as, for example, the amino acid of Formula IV,wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.In certain embodiments, n is 2, 3, 4, or 5, in which case, the aminoacid is Dab, Orn, Lys, or homoLys respectively.

The alpha, alpha-disubstituted amino acid of the analog of theseembodiments may be any alpha, alpha-disubstituted amino acid, including,but not limited to, amino iso-butyric acid (Aib), an amino aciddisubstituted with the same or a different group selected from methyl,ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In certain embodiments, thealpha, alpha-disubstituted amino acid is Aib.

In accordance with these embodiments, the analog can comprise, forexample, the amino acid sequence of any of SEQ ID NOs: 1099-1165.

In yet other exemplary embodiments, the analog of glucagon (SEQ ID NO:1001) having GIP agonist activity comprises:

(a) an amino acid modification at position 1 that confers GIP agonistactivity, and

(b) an extension of about 1 to about 21 amino acids C-terminal to theamino acid at position 29, wherein at least one of the amino acids ofthe extension is acylated or alkylated,

wherein the EC₅₀ of the analog for GIP receptor activation is about 10nM or less.

In some embodiments, the acylated or alkylated amino acid is an aminoacid of Formula I, II, or III. In more specific embodiments, the aminoacid of Formula I is Dab, Orn, Lys, or homoLys. Also, in someembodiments, the extension of about 1 to about 21 amino acids comprisesthe amino acid sequence of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS(SEQ ID NO: 1096), wherein X is any amino acid, or GPSSGAPPPK (SEQ IDNO: 1170) or XGPSSGAPPPK (SEQ ID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO:1172), wherein X is Gly or a small, aliphatic or non-polar or slightlypolar amino acid. In some embodiments, the about 1 to about 21 aminoacids may comprise sequences containing one or more conservativesubstitutions relative to SEQ ID NO: 1095, 1096, 1170, 1171 or 1172. Insome embodiments, the acylated or alkylated amino acid is located atposition 37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extendedanalog. In certain embodiments, the acylated or alkylated amino acid islocated at position 40 of the C-terminally extended analog.

In some embodiments, the analog having GIP agonist activity furthercomprises amino acid modifications at one, two or all of positions 27,28 and 29, e.g., amino acid modifications at position 27 and/or 28.

In any of the above exemplary embodiments, the amino acid modificationat position 1 that confers GIP agonist activity can be a substitution ofHis with an amino acid lacking an imidazole side chain. The amino acidmodification at position 1 can, for example, be a substitution of Hiswith a large, aromatic amino acid. In some embodiments, the large,aromatic amino acid is any of those described herein, including, forexample, Tyr.

In certain aspects, the analog does not comprise an amino acidmodification at position 1 which modification confers GIP agonistactivity. In some aspects, the amino acid at position 1 is not a large,aromatic amino acid, e.g., Tyr. In some embodiments, the amino acid atposition 1 is an amino acid comprising an imidazole ring, e.g., His,analogs of His. In certain embodiments, the analog is not any of thecompounds disclosed in International Patent Application Publication No.WO 2010/011439. In certain aspects, the analog comprises the amino acidsequence of any of SEQ ID NOs: 1263-1275.

Also, with regard to the above exemplary embodiments, amino acidmodifications at one, two, or all of positions 27, 28, and 29 can be anyof the modifications at these positions described herein. For example,the Met at position 27 can be substituted with a large aliphatic aminoacid, optionally Leu, the Asn at position 28 can be substituted with asmall aliphatic amino acid, optionally Ala, and/or the Thr at position29 can be substituted with a small aliphatic amino acid, optionally Gly.Alternatively, the analog can comprise such amino acid modifications atposition 27 and/or 28.

The analog of the above exemplary embodiments can further comprise 1-9or 1-6 further, additional amino acid modifications, e.g. 1, 2, 3, 4, 5,6, 7, 8 or 9 further amino acid modifications, such as, for example, anyof the modifications described herein which increase or decrease theactivity at any of the GIP, GLP-1, and glucagon receptors, improvesolubility, improve duration of action or half-life in circulation,delay the onset of action, or increase stability. The analog can furthercomprise, for example, an amino acid modification at position 12,optionally, a substitution with Ile, and/or amino acid modifications atpositions 17 and 18, optionally substitution with Q at position 17 and Aat position 18, and/or an addition of GPSSGAPPPS (SEQ ID NO: 1095) orXGPSSGAPPPS (SEQ ID NO: 1096), or sequences containing one or moreconservative substitutions relative to SEQ ID NO: 1095 or 1096, to theC-terminus. The analog can comprise one or more of the followingmodifications:

(i) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,N-methyl-Ser, Aib, Val, or α-amino-N-butyric acid;

(ii) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe, orVal;

(iii) Linkage of an acyl group to a Lys at position 10;

(iv) Lys at position 12 substituted with Arg;

(v) Ser at position 16 substituted with Glu, Gln, homoglutamic acid,homocysteic acid, Thr, Gly, or Aib;

(vi) Arg at position 17 substituted with Gln;

(vii) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;

(viii) Gln at position 20 substituted with Ala, Ser, Thr, Lys,Citrulline, Arg, Orn, or Aib;

(ix) Asp at position 21 substituted with Glu, homoglutamic acid,homocysteic acid;

(x) Val at position 23 substituted with Ile;

(xi) Gln at position 24 substituted with Asn, Ala, Ser, Thr, or Aib; and

(xii) a conservative substitution at any of positions 2, 5, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.

The analog in some embodiments comprise a combination of themodifications (i) through (xii). Alternatively or additionally, theanalog can comprise an amino acid modification at position 3 (e.g., anamino acid substitution of Gln with Glu), wherein the analog has lessthan 1% of the activity of glucagon at the glucagon receptor.Alternatively or additionally, the analog can comprise an amino acidmodification at position 7 (e.g., an amino acid substitution of Thr withan amino acid lacking a hydroxyl group, e.g., Abu or Ile), wherein theanalog has less than about 10% of the activity of GLP-1 at the GLP-1receptor.

With regard to the exemplary embodiments, the analog can be covalentlylinked to a hydrophilic moiety. In some embodiments, the analog iscovalently linked to the hydrophilic moiety at any of amino acidpositions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. In certainembodiments, the analog comprises a C-terminal extension (e.g., an aminoacid sequence of SEQ ID NO: 1095) and an addition of an amino acidcomprising the hydrophilic moiety, such that the hydrophilic moiety iscovalently linked to the analog at position 40.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. TheLys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an aminoacid that is native to the glucagon sequence (SEQ ID NO: 1001) or it maybe an amino acid which is replacing a native amino acid of SEQ ID NO:1001. In some embodiments, wherein the hydrophilic moiety is attached toa Cys, the linkage to the hydrophilic moiety can comprise the structure

With regard to the analogs comprising a hydrophilic moiety, thehydrophilic moiety may be any of those described herein. See, e.g., theteachings under the section “Linkage of hydrophilic moieties.” In someembodiments, the hydrophilic moiety is a polyethylene glycol (PEG). ThePEG in certain embodiments has a molecular weight of about 1,000 Daltonsto about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000Daltons.

With regard to the exemplary embodiments, the analog can comprise amodified amino acid in which the side chain is covalently linked to anacyl or alkyl group (e.g., an acyl or alkyl group which is non-native toa naturally-occurring amino acid). The acylated or alkylated analog canbe in accordance with acylated or alkylated peptides described in thesection “Acylation and alkylation.” In some embodiments, the acyl groupis a C4 to a C30 fatty acyl group, such as, for example, a C10 fattyacyl or alkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acylor alkyl group, a C16 fatty acyl or alkyl group, a C18 fatty acyl oralkyl group, a C20 acyl or alkyl group, or a C22 acyl or alkyl group.The acyl or alkyl group may be covalently attached to any amino acid ofthe analog, including, but not limited to the amino acid at position 10or 40, or the C-terminal amino acid. In certain embodiments, the analogcomprises a C-terminal extension (e.g., an amino acid sequence of SEQ IDNO: 1095) and an addition of an amino acid comprising the acyl or alkylgroup, such that the acyl or alkyl group is covalently linked to theanalog at position 40. In some embodiments, the acyl or alkyl group iscovalently linked to the side chain of an amino acid of Formula I, II,or III, e.g., a Lys residue. The acyl or alkyl group may be covalentlylinked to an amino acid which is native to the glucagon sequence (SEQ IDNO: 1001) or may be linked to an amino acid which is added to thesequence of SEQ ID NO: 1001 or to the sequence of SEQ ID NO: 1001followed by SEQ ID NO: 1095 (at the N- or C-terminus) or may be linkedto an amino acid which replaces a native amino acid, e.g., the Tyr atposition 10 of SEQ ID NO: 1001.

In the above exemplary embodiments, wherein the analog comprises an acylor alkyl group, the analog may be attached to the acyl or alkyl groupvia a spacer, as described herein. The spacer, for example, may be 3 to10 atoms in length and may be, for instance, an amino acid (e.g.,6-amino hexanoic acid, any amino acid described herein), a dipeptide(e.g., Ala-Ala, βAla-βAla, Leu-Leu, Pro-Pro, γ-Glu-γ-Glu), a tripeptide,or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects,the total length of the spacer and the acyl or alkyl group is about 14to about 28 atoms. In some embodiments, the amino acid spacer is notγ-Glu. In some embodiments, the dipeptide spacer is not γ-Glu-γ-Glu.

In still further exemplary embodiments, the analog of glucagon havingGIP agonist activity comprises the amino acid sequence according to anyone of SEQ ID NOs: 1227, 1228, 1229 or 1230 that further comprises thefollowing modifications:

(a) optionally, an amino acid modification at position 1 that confersGIP agonist activity,

(b) an extension of about 1 to about 21 amino acids C-terminal to theamino acid at position 29, wherein at least one of the amino acids ofthe extension is acylated or alkylated, and

(d) up to 6 further amino acid modifications,

wherein the EC₅₀ of the analog for GIP receptor activation is about 10nM or less.

In some aspects, the acylated or alkylated amino acid is an amino acidof Formula I, II, or III. In more specific embodiments, the amino acidof Formula I is Dab, Orn, Lys, or homoLys. Also, in some embodiments,the about 1 to about 21 amino acids comprises the amino acid sequence ofGPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQ ID NO: 1096), wherein Xis any amino acid, or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSSGAPPPK (SEQID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO: 1172), wherein Xis Gly or asmall, aliphatic or non-polar or slightly polar amino acid. In someembodiments, the about 1 to about 21 amino acids may comprise sequencescontaining one or more conservative substitutions relative to SEQ ID NO:1095, 1096, 1170, 1171 or 1172. In some embodiments, the acylated oralkylated amino acid is located at position 37, 38, 39, 40, 41, 42, or43 of the C-terminally-extended analog. In certain embodiments, theacylated or alkylated amino acid is located at position 40 of theC-terminally extended analog.

In any of the above exemplary embodiments, the amino acid at position 1that confers GIP agonist activity can be an amino acid lacking animidazole side chain. The amino acid at position 1 can, for example, bea large, aromatic amino acid. In some embodiments, the large, aromaticamino acid is any of those described herein, including, for example,Tyr.

The analog of the above exemplary embodiments can further comprise 1-6further amino acid modifications, such as, for example, any of themodifications described herein which increase or decrease the activityat any of the GIP, GLP-1, and glucagon receptors, improve solubility,improve duration of action or half-life in circulation, delay the onsetof action, or increase stability.

In certain aspects, glucagon analogs described in the above exemplaryembodiment, comprise further amino acid modifications at one, two or allof positions 27, 28 and 29. Modifications at these positions can be anyof the modifications described herein relative to these positions. Forexample, relative to SEQ ID NO: 1227, 1228, 1229 or 1230, position 27can be substituted with a large aliphatic amino acid (e.g., Leu, Ile ornorleucine) or Met, position 28 can be substituted with another smallaliphatic amino acid (e.g., Gly or Ala) or Asn, and/or position 29 canbe substituted with another small aliphatic amino acid (e.g., Ala orGly) or Thr. Alternatively, the analog can comprise such amino acidmodifications at position 27 and/or 28.

The analog can further comprise one or more of the following additionalmodifications:

(i) the amino acid at position 2 is any one of D-Ser, Ala, D-Ala, Gly,N-methyl-Ser, Aib, Val, or α-amino-N-butyric acid;

(ii) the amino acid at position 10 is Tyr, Trp, Lys, Orn, Glu, Phe, orVal;

(iii) linkage of an acyl group to a Lys at position 10;

(iv) the amino acid at position 12 is Ile, Lys or Arg;

(v) the amino acid at position 16 is any one of Ser, Glu, Gln,homoglutamic acid, homocysteic acid, Thr, Gly, or Aib;

(vi) the amino acid at position 17 is Gln or Arg;

(vii) the amino acid at position 18 is any one of Ala, Arg, Ser, Thr, orGly;

(viii) the amino acid at position 20 is any one of Ala, Ser, Thr, Lys,Citrulline, Arg, Orn, or Aib or another alpha, alpha-disubstituted aminoacid;

(ix) the amino acid at position 21 is any one of Glu, Asp, homoglutamicacid, homocysteic acid;

(x) the amino acid at position 23 is Val or Ile;

(xi) the amino acid at position 24 is any one of Gln, Asn, Ala, Ser,Thr, or Aib; and

(xii) one or more conservative substitutions at any of positions 2, 5,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.

The analog in some embodiments comprise a combination of themodifications (i) through (xii). Alternatively or additionally, theanalog can comprise an amino acid modification at position 3 (e.g., anamino acid substitution of Gln with Glu), wherein the analog has lessthan 1% of the activity of glucagon at the glucagon receptor.Alternatively or additionally, the analog can comprise an amino acidmodification at position 7 (e.g., an amino acid substitution of Thr withan amino acid lacking a hydroxyl group, e.g., Abu or Ile), wherein theanalog has less than about 10% of the activity of GLP-1 at the GLP-1receptor.

With regard to the exemplary embodiments, the analog can be covalentlylinked to a hydrophilic moiety. In some embodiments, the analog iscovalently linked to the hydrophilic moiety at any of amino acidpositions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. In certainembodiments, the analog comprises a hydrophilic moiety covalently linkedto the analog at position 24.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. TheLys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an aminoacid that is native to SEQ ID NO: 1001, 1227, 1228, 1229 or 1230 or itmay be a substituted amino acid. In some embodiments, wherein thehydrophilic moiety is linked to a Cys, the linkage may comprise thestructure

With regard to the analogs comprising a hydrophilic moiety, thehydrophilic moiety may be any of those described herein. See, e.g., theteachings under the section “Linkage of hydrophilic moieties.” In someembodiments, the hydrophilic moiety is a polyethylene glycol (PEG). ThePEG in certain embodiments has a molecular weight of about 1,000 Daltonsto about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000Daltons.

With regard to the exemplary embodiments, the analog can comprise amodified amino acid within the C-terminal extension in which the sidechain is covalently linked to an acyl or alkyl group. The acylated oralkylated analog can be in accordance with acylated or alkylatedpeptides described in the section “Acylation and alkylation.” In someembodiments, the acyl group is a C4 to a C30 fatty acyl group, such as,for example, a C10 fatty acyl or alkyl group, a C12 fatty acyl or alkylgroup, a C14 fatty acyl or alkyl group, a C16 fatty acyl or alkyl group,a C18 fatty acyl or alkyl group, a C20 acyl or alkyl group, or a C22acyl or alkyl group. The acyl or alkyl group may be covalently attachedto any amino acid of the analog, including, but not limited to the aminoacid at position 10 or 40, or the C-terminal amino acid. In someembodiments, the acyl or alkyl group is covalently linked to the sidechain of an amino acid of Formula I, II, or III, e.g., a Lys residue.The acyl or alkyl group is covalently linked to an amino acid which isnative to SEQ ID NO: 1001, 1227, 1228, 1229 or 1230 or it may be linkedto a substituted amino acid. The acyl or alkyl group is covalentlylinked to an amino acid which is native to SEQ ID NO: 1095, 1096, 1171or 1172, or it may be linked to a substituted amino acid.

In the above exemplary embodiments, wherein the analog comprises an acylor alkyl group, the analog may be attached to the acyl or alkyl groupvia a spacer, as described herein. The spacer, for example, may be 3 to10 atoms in length and may be, for instance, an amino acid (e.g.,6-amino hexanoic acid, any amino acid described herein), a dipeptide(e.g., Ala-Ala, βAla-βAla, Leu-Leu, Pro-Pro, γ-Glu-γ-Glu), a tripeptide,or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects,the total length of the spacer and the acyl or alkyl group is about 14to about 28 atoms. In some embodiments, the amino acid spacer is notγ-Glu. In some embodiments, the dipeptide spacer is not γ-Glu-γ-Glu.

In some very specific embodiments, an analog of the invention comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:1099-1141, 1144-1164, 1166, 1192-1207, 1209-1221 and 1223 or selectedfrom the group consisting of SEQ ID NOs: 1167-1169, 1173-1178 and 1225.

Further, specific examples of analogs of the invention include but arenot limited to, any of those referenced in Tables 1-3.

In still further exemplary embodiments, the analog of glucagon havingGIP agonist activity comprises an acyl or alkyl group (e.g., an acyl oralkyl group which is non-native to a naturally occurring amino acid),wherein the acyl or alkyl group is attached to a spacer, wherein (i) thespacer is attached to the side chain of the amino acid at position 10 ofthe analog; or (ii) the analog comprises an extension of 1 to 21 aminoacids C-terminal to the amino acid at position 29 and the spacer isattached to the side chain of an amino acid corresponding to one ofpositions 37-43 relative to SEQ ID NO: 1001, wherein the EC₅₀ of theanalog for GIP receptor activation is about 10 nM or less.

In such embodiments, the analog may comprise an amino acid sequence ofSEQ ID NO: 1001 with (i) an amino acid modification at position 1 thatconfers GIP agonist activity, (ii) amino acid modifications at one, two,or all of positions 27, 28, and 29, (iii) at least one of:

(A) the analog comprises a lactam bridge between the side chains ofamino acids at positions i and i+4 or between the side chains of aminoacids at positions j and j+3, wherein i is 12, 13, 16, 17, 20 or 24, andwherein j is 17;

(B) one, two, three, or all of the amino acids at positions 16, 20, 21,and 24 of the analog is substituted with an α,α-disubstituted aminoacid; or

(C) the analog comprises (i) an amino acid substitution of Ser atposition 16 with an amino acid of Formula IV:

wherein n is 1 to 7, wherein each of R1 and R2 is independently selectedfrom the group consisting of H, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl), wherein R₇ is H or OH, and the side chain ofthe amino acid of Formula IV comprises a free amino group; and (ii) anamino acid substitution of the Gln at position 20 with an alpha,alpha-disubstituted amino acid.

and (iv) up to 6 further amino acid modifications.

The alpha, alpha-disubstituted amino acid of the analog of theseembodiments may be any alpha, alpha-disubstituted amino acid, including,but not limited to, amino iso-butyric acid (Aib), an amino aciddisubstituted with the same or a different group selected from methyl,ethyl, propyl, and n-butyl, or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). In certain embodiments, thealpha, alpha-disubstituted amino acid is Aib.

The amino acid of Formula IV of the analog of these embodiments may beany amino acid, such as, for example, the amino acid of Formula IV,wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.In certain embodiments, n is 2, 3, 4, or 5, in which case, the aminoacid is Dab, Orn, Lys, or homoLys respectively.

In any of the above exemplary embodiments, the amino acid modificationat position 1 that confers GIP agonist activity can be a substitution ofHis with an amino acid lacking an imidazole side chain. The amino acidmodification at position 1 can, for example, be a substitution of Hiswith a large, aromatic amino acid. In some embodiments, the large,aromatic amino acid is any of those described herein, including, forexample, Tyr.

Also, with regard to the above exemplary embodiments, amino acidmodifications at one, two, or all of positions 27, 28, and 29 can be anyof the modifications at these positions described herein. For example,the Met at position 27 can be substituted with a large aliphatic aminoacid, optionally Leu, the Asn at position 28 can be substituted with asmall aliphatic amino acid, optionally Ala, and/or the Thr at position29 can be substituted with a small aliphatic amino acid, optionally Gly.Alternatively, the analog can comprise such amino acid modifications atposition 27 and/or 28.

The analog of the above exemplary embodiments can further comprise 1-9or 1-6 further, additional amino acid modifications, e.g. 1, 2, 3, 4, 5,6, 7, 8 or 9 further amino acid modifications, such as, for example, anyof the modifications described herein which increase or decrease theactivity at any of the GIP, GLP-1, and glucagon receptors, improvesolubility, improve duration of action or half-life in circulation,delay the onset of action, or increase stability. The analog can furthercomprise, for example, an amino acid modification at position 12,optionally, a substitution with Ile, and/or amino acid modifications atpositions 17 and 18, optionally substitution with Q at position 17 and Aat position 18, and/or an addition of GPSSGAPPPS (SEQ ID NO: 1095) orXGPSSGAPPPS (SEQ ID NO: 1096), or sequences containing one or moreconservative substitutions relative to SEQ ID NO: 1095 or 1096, to theC-terminus. The analog can comprise one or more of the followingmodifications:

(i) Ser at position 2 substituted with D-Ser, Ala, D-Ala, Gly,N-methyl-Ser, Aib, Val, or α-amino-N-butyric acid;

(ii) Tyr at position 10 substituted with Trp, Lys, Orn, Glu, Phe, orVal;

(iii) Linkage of an acyl group to a Lys at position 10;

(iv) Lys at position 12 substituted with Arg;

(v) Ser at position 16 substituted with Glu, Gln, homoglutamic acid,homocysteic acid, Thr, Gly, Lys, or Aib;

(vi) Arg at position 17 substituted with Gln;

(vii) Arg at position 18 substituted with Ala, Ser, Thr, or Gly;

(viii) Gln at position 20 substituted with Ala, Ser, Thr, Lys,Citrulline, Arg, Orn, or

Aib;

(ix) Asp at position 21 substituted with Glu, homoglutamic acid,homocysteic acid;

(x) Val at position 23 substituted with Ile;

(xi) Gln at position 24 substituted with Asn, Ala, Ser, Thr, or Aib; and

(xii) a conservative substitution at any of positions 2, 5, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28, and 29.

The analog in some embodiments comprise a combination of themodifications (i) through (xii). Alternatively or additionally, theanalog can comprise an amino acid modification at position 3 (e.g., anamino acid substitution of Gln with Glu), wherein the analog has lessthan 1% of the activity of glucagon at the glucagon receptor.Alternatively or additionally, the analog can comprise an amino acidmodification at position 7 (e.g., an amino acid substitution of Thr withan amino acid lacking a hydroxyl group, e.g., Abu or Ile), a deletion ofthe amino acid(s)C-terminal to the amino acid at position 27 or 28,yielding a 27- or 28-amino acid peptide, or a combination thereof,wherein the analog has less than about 10% of the activity of GLP-1 atthe GLP-1 receptor.

With regard to the exemplary embodiments, the analog can be covalentlylinked to a hydrophilic moiety. In some embodiments, the analog iscovalently linked to the hydrophilic moiety at any of amino acidpositions 16, 17, 20, 21, 24, 29, 40, or the C-terminus. In certainembodiments, the analog comprises a C-terminal extension (e.g., an aminoacid sequence of SEQ ID NO: 1095) and an addition of an amino acidcomprising the hydrophilic moiety, such that the hydrophilic moiety iscovalently linked to the analog at position 40.

In some embodiments, the hydrophilic moiety is covalently linked to aLys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. TheLys, Cys, Orn, homocysteine, or acetyl-phenylalanine may be an aminoacid that is native to the glucagon sequence (SEQ ID NO: 1001) or it maybe an amino acid which is replacing a native amino acid of SEQ ID NO:1001. In some embodiments, wherein the hydrophilic moiety is attached toa Cys, the linkage to the hydrophilic moiety can comprise the structure

With regard to the analogs comprising a hydrophilic moiety, thehydrophilic moiety may be any of those described herein. See, e.g., theteachings under the section “Linkage of hydrophilic moieties.” In someembodiments, the hydrophilic moiety is a polyethylene glycol (PEG). ThePEG in certain embodiments has a molecular weight of about 1,000 Daltonsto about 40,000 Daltons, e.g., about 20,000 Daltons to about 40,000Daltons.

In the exemplary embodiments, wherein the analog comprises an acyl oralkyl group, which is attached to the analog via a spacer, the spacercan be any spacer as described herein. The spacer, for example, may be 3to 10 atoms in length and may be, for instance, an amino acid (e.g.,6-amino hexanoic acid, any amino acid described herein), a dipeptide(e.g., Ala-Ala, βAla-βAla, Leu-Leu, Pro-Pro, γ-Glu-γ-Glu), a tripeptide,or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects,the total length of the spacer and the acyl or alkyl group is about 14to about 28 atoms. In some embodiments, the amino acid spacer is notγ-Glu. In some embodiments, the dipeptide spacer is not γ-Glu-γ-Glu.

The acyl or alkyl group is any acyl or alkyl group as described herein,such as an acyl or alkyl group which is non-native to a naturallyoccurring amino acid. The acyl or alkyl group in some embodiments is aC4 to C30 fatty acyl group, such as, for example, a C10 fatty acyl oralkyl group, a C12 fatty acyl or alkyl group, a C14 fatty acyl or alkylgroup, a C16 fatty acyl or alkyl group, a C18 fatty acyl or alkyl group,a C20 acyl or alkyl group, or a C22 acyl or alkyl group, or a C4 to C30alkyl group. In specific embodiments, the acyl group is a C12 to C18fatty acyl group (e.g., a C14 or C16 fatty acyl group).

In some embodiments, the extension of about 1 to about 21 amino acidsC-terminal to the amino acid at position 29 of the analog comprises theamino acid sequence of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSSGAPPPS (SEQID NO: 1096), wherein X is any amino acid, or GPSSGAPPPK (SEQ ID NO:1170) or XGPSSGAPPPK (SEQ ID NO: 1171) or XGPSSGAPPPSK (SEQ ID NO:1172), wherein X is Gly or a small, aliphatic or non-polar or slightlypolar amino acid. In some embodiments, the about 1 to about 21 aminoacids may comprise sequences containing one or more conservativesubstitutions relative to SEQ ID NO: 1095, 1096, 1170, 1171 or 1172. Insome embodiments, the acylated or alkylated amino acid is located atposition 37, 38, 39, 40, 41, 42, or 43 of the C-terminally-extendedanalog. In certain embodiments, the acylated or alkylated amino acid islocated at position 40 of the C-terminally extended analog.

The GIP agonist may be a peptide comprising the amino acid sequence ofany of the amino acid sequences, e.g., SEQ ID NOs: 1005-1094, optionallywith up to 1, 2, 3, 4, or 5 further modifications that retain GIPagonist activity. In certain embodiments, the GIP agonist comprises theamino acids of any of SEQ ID NOs: 1099-1275.

Class 3 Glucagon Related Peptides

In certain embodiments, the glucagon related peptide is a Class 3glucagon related peptide, which is described herein and in InternationalPatent Application Publication Nos. WO 2009/155258, WO 2008/101017, andU.S. Provisional Application No. 61/288,248 (filed on Dec. 18, 2009) thecontents of which are incorporated by reference in their entirety.

Some of the biological sequences referenced in the following section(SEQ ID NOs: 1-656) relating to Class 3 glucagon related peptides arecorrespond to SEQ ID NOs: 1-656 in International Patent ApplicationPublication No. WO 2009/155258.

Activity

The Class 3 glucagon related peptide can be a peptide that exhibitsincreased activity at the glucagon receptor, and in further embodimentsexhibits enhanced biophysical stability and/or aqueous solubility. Inaddition, in some embodiments, the Class 3 glucagon related peptide haslost native glucagon's selectivity for the glucagon receptor verses theGLP-1 receptor, and thus represents co-agonists of those two receptors.Selected amino acid modifications within the Class 3 glucagon relatedpeptide can control the relative activity of the peptide at the GLP-1receptor verses the glucagon receptor. Thus, the Class 3 glucagonrelated peptide can be a glucagon/GLP-1 co-agonist that has higheractivity at the glucagon receptor versus the GLP-1 receptor, aglucagon/GLP-1 co-agonist that has approximately equivalent activity atboth receptors, or a glucagon/GLP-1 co-agonist that has higher activityat the GLP-1 receptor versus the glucagon receptor. The latter categoryof co-agonist can be engineered to exhibit little or no activity at theglucagon receptor, and yet retain ability to activate the GLP-1 receptorwith the same or better potency than native GLP-1. Any of theseco-agonists may also include modifications that confer enhancedbiophysical stability and/or aqueous solubility.

Modifications of the Class 3 glucagon related peptide can be made toproduce a glucagon peptide having anywhere from at least about 1%(including at least about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%,60%, 75%, 100%, 125%, 150%, 175%) to about 200% or higher activity atthe GLP-1 receptor relative to native GLP-1 and anywhere from at leastabout 1% (including about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%,60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%) toabout 500% or higher activity at the glucagon receptor relative tonative glucagon. The amino acid sequence of native glucagon is SEQ IDNO: 1, the amino acid sequence of GLP-1(7-36)amide is SEQ ID NO: 52, andthe amino acid sequence of GLP-1(7-37) acid is SEQ ID NO: 50. Inexemplary embodiments, a Class 3 glucagon related peptide may exhibit atleast 10% of the activity of native glucagon at the glucagon receptorand at least 50% of the activity of native GLP-1 at the GLP-1 receptor,or at least 40% of the activity of native glucagon at the glucagonreceptor and at least 40% of the activity of native GLP-1 at the GLP-1receptor, or at least 60% of the activity of native glucagon at theglucagon receptor and at least 60% of the activity of native GLP-1 atthe GLP-1 receptor.

Selectivity of a Class 3 glucagon related peptide for the glucagonreceptor versus the GLP-1 receptor can be described as the relativeratio of glucagon/GLP-1 activity (the peptide's activity at the glucagonreceptor relative to native glucagon, divided by the peptide's activityat the GLP-1 receptor relative to native GLP-1). For example, a Class 3glucagon related peptide that exhibits 60% of the activity of nativeglucagon at the glucagon receptor and 60% of the activity of nativeGLP-1 at the GLP-1 receptor has a 1:1 ratio of glucagon/GLP-1 activity.Exemplary ratios of glucagon/GLP-1 activity include about 1:1, 1.5:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, or about 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.5. As an example, a glucagon/GLP-1activity ratio of 10:1 indicates a 10-fold selectivity for the glucagonreceptor versus the GLP-1 receptor. Similarly, a GLP-1/glucagon activityratio of 10:1 indicates a 10-fold selectivity for the GLP-1 receptorversus the glucagon receptor.

In some embodiments, the Class 3 glucagon related peptides have about10% or less of the activity of native glucagon at the glucagon receptor,e.g. about 1-10%, or about 0.1-10%, or greater than about 0.1% but lessthan about 10%, while exhibiting at least 20% of the activity of GLP-1at the GLP-1 receptor. For example, exemplary Class 3 glucagon relatedpeptides described herein have about 0.5%, about 1% or about 7% of theactivity of native glucagon, while exhibiting at least 20% of theactivity of GLP-1 at the GLP-1 receptor.

The Class 3 glucagon related peptide can be a glucagon peptide withincreased or decreased activity at the glucagon receptor, or GLP-1receptor, or both. The Class 3 glucagon related peptide can be aglucagon peptide with altered selectivity for the glucagon receptorversus the GLP-1 receptor.

Thus, as disclosed herein high potency Class 3 glucagon related peptidesare provided that also exhibit improved solubility and/or stability. Anexemplary high potency Class 3 glucagon related peptide exhibits atleast about 200% of the activity of native glucagon at the glucagonreceptor, and optionally is soluble at a concentration of at least 1mg/mL at a pH between 6 and 8, or between 6 and 9, or between 7 and 9(e.g. pH 7), and optionally retains at least 95% of the original peptide(e.g. 5% or less of the original peptide is degraded or cleaved) after24 hours at 25° C. As another example, an exemplary Class 3 glucagonrelated peptide exhibits greater than about 40% or greater than about60% activity at both the glucagon and the GLP-1 receptors (at a ratiobetween about 1:3 and 3:1, or between about 1:2 and 2:1), is optionallysoluble at a concentration of at least 1 mg/mL at a pH between 6 and 8or between 6 and 9, or between 7 and 9 (e.g. pH 7), and optionallyretains at least 95% of the original peptide after 24 hours at 25° C.Another exemplary Class 3 glucagon related peptide exhibits about 175%or more of the activity of native glucagon at the glucagon receptor andabout 20% or less of the activity of native GLP-1 at the GLP-1 receptor,is optionally soluble at a concentration of at least 1 mg/mL at a pHbetween 6 and 8 or between 6 and 9, or between 7 and 9 (e.g. pH 7), andoptionally retains at least 95% of the original peptide after 24 hoursat 25° C. Yet another exemplary Class 3 glucagon related peptideexhibits about 10% or less of the activity of native glucagon at theglucagon receptor and at least about 20% of the activity of native GLP-1at the GLP-1 receptor, is optionally soluble at a concentration of atleast 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7and 9 (e.g. pH 7), and optionally retains at least 95% of the originalpeptide after 24 hours at 25° C. Yet another exemplary Class 3 glucagonrelated peptide exhibits about 10% or less but above 0.1%, 0.5% or 1% ofthe activity of native glucagon at the glucagon receptor and at leastabout 50%, 60%, 70%, 80%, 90% or 100% or more of the activity of nativeGLP-1 at the GLP-1 receptor, is optionally soluble at a concentration ofat least 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between7 and 9 (e.g. pH 7), and optionally retains at least 95% of the originalpeptide after 24 hours at 25° C. In some embodiments, such Class 3glucagon related peptides retain at least 22, 23, 24, 25, 26, 27 or 28of the naturally occurring amino acids at the corresponding positions innative glucagon (e.g. have 1-7, 1-5 or 1-3 modifications relative tonaturally occurring glucagon).

Modifications Affecting Glucagon Activity

Increased activity at the glucagon receptor is provided by an amino acidmodification at position 16 of native glucagon (SEQ ID NO: 1). In someembodiments, the Class 3 glucagon related peptide is a glucagon agonistthat has been modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1) to enhance the peptide's potency at the glucagonreceptor. The normally occurring serine at position 16 of nativeglucagon (SEQ ID NO: 1) can be substituted with select acidic aminoacids to enhance the potency of glucagon, in terms of its ability tostimulate cAMP synthesis in a validated in vitro model assay (seeExample 2). More particularly, this substitution enhances the potency ofthe analog at least 2-fold, 4-fold, 5-fold, and up to 10-fold greater atthe glucagon receptor. This substitution also enhances the analog'sactivity at the GLP-1 receptor at least 5-fold, 10-fold, or 15-foldrelative to native glucagon, but selectivity is maintained for theglucagon receptor over the GLP-1 receptor.

By way of nonlimiting example, such enhanced potency can be provided bysubstituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms. In accordance withsome embodiments, the serine residue at position 16 of native glucagonis substituted with an amino acid selected from the group consisting ofglutamic acid, glutamine, homoglutamic acid, homocysteic acid,threonine, or glycine. In accordance with some embodiments, the serineresidue at position 16 of native glucagon is substituted with an aminoacid selected from the group consisting of glutamic acid, glutamine,homoglutamic acid and homocysteic acid, and in some embodiments theserine residue is substituted with glutamic acid.

In some embodiments, the enhanced potency Class 3 glucagon relatedpeptide comprises a peptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or a glucagon agonist analog ofSEQ ID NO: 5. In accordance with some embodiments, a Class 3 glucagonrelated peptide having enhanced potency at the glucagon receptorrelative to wild type glucagon is provided wherein the peptide comprisesthe sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:10, wherein the glucagon peptide retains its selectivity for theglucagon receptor relative to the GLP-1 receptors. In some embodiments,the Class 3 glucagon related peptide having enhanced specificity for theglucagon receptor comprises the peptide of SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10 or a glucagon agonist analog thereof, wherein the carboxyterminal amino acid retains its native carboxylic acid group. Inaccordance with some embodiments, a Class 3 glucagon related peptidecomprises the sequence ofNH₂—His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH(SEQ ID NO: 10), wherein the peptide exhibits approximately fivefoldenhanced potency at the glucagon receptor, relative to native glucagonas measured by the in vitro cAMP assay of Example 2.

Glucagon receptor activity can be reduced, maintained, or enhanced by anamino acid modification at position 3, e.g. substitution of thenaturally occurring glutamine at position 3. In some embodiments,substitution of the amino acid at position 3 with an acidic, basic, orhydrophobic amino acid (glutamic acid, ornithine, norleucine) has beenshown to substantially reduce or destroy glucagon receptor activity. Theanalogs that are substituted with, for example, glutamic acid,ornithine, or norleucine have about 10% or less of the activity ofnative glucagon at the glucagon receptor, e.g. about 1-10%, or about0.1-10%, or greater than about 0.1% but less than about 10%, whileexhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.For example, exemplary analogs described herein have about 0.5%, about1% or about 7% of the activity of native glucagon, while exhibiting atleast 20% of the activity of GLP-1 at the GLP-1 receptor. In particular,any of the Class 3 glucagon related peptides, including glucagonanalogs, glucagon agonist analogs, glucagon co-agonists, andglucagon/GLP-1 co-agonist molecules, described herein may be modified tocontain a modification at position 3, e.g., Gln substituted with Glu, toproduce a peptide with high selectivity, e.g., tenfold selectivity, forthe GLP-1 receptor as compared to the selectivity for the glucagonreceptor.

In another embodiment, the naturally occurring glutamine at position 3of any of the Class 3 glucagon peptides can be substituted with aglutamine analog without a substantial loss of activity at the glucagonreceptor, and in some cases, with an enhancement of glucagon receptoractivity, as described herein. In specific embodiments, the amino acidat position 3 is substituted with Dab(Ac). For example, glucagonagonists can comprise the amino acid sequence of SEQ ID NO: 595, SEQ IDNO: 601 SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, and SEQ ID NO:606.

It was observed that modifications at position 2 (e.g. Aib at position2) and in some cases modifications at position 1 may reduce glucagonactivity. This reduction in glucagon activity can be restored bystabilizing the alpha-helix in the C-terminal portion of glucagon, e.g.through means described herein, for example, through a covalent bondbetween the side chains of the amino acids at positions “i” and “i+4”,e.g., 12 and 16, 16 and 20, or 20 and 24. In some embodiments, thiscovalent bond is a lactam bridge between a glutamic acid at position 16and a lysine at position 20. In some embodiments, this covalent bond isan intramolecular bridge other than a lactam bridge. For example,suitable covalent bonding methods include any one or more of olefinmetathesis, lanthionine-based cyclization, disulfide bridge or modifiedsulfur-containing bridge formation, the use of a, co-diaminoalkanetethers, the formation of metal-atom bridges, and other means of peptidecyclization.

Modifications Affecting GLP-1 Activity

Enhanced activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester. In some embodiments, these Class 3glucagon related peptides comprise a sequence of SEQ ID NO: 20, whereinthe carboxy terminal amino acid has an amide group in place of thecarboxylic acid group found on the native amino acid. These Class 3glucagon related peptides have strong activity at both the glucagon andGLP-1 receptors and thus act as co-agonists at both receptors. Inaccordance with some embodiments, the Class 3 glucagon related peptideis a glucagon and GLP-1 receptor co-agonist, wherein the peptidecomprises the sequence of SEQ ID NO: 20, wherein the amino acid atposition 28 is Asn or Lys and the amino acid at position 29 is Thr-amide

Increased activity at the GLP-1 receptor is provided by modificationsthat stabilize the alpha helix in the C-terminal portion of glucagon(e.g. around residues 12-29).

In some embodiments, such modifications permit formation of anintramolecular bridge between the side chains of two amino acids thatare separated by three intervening amino acids (i.e., an amino acid atposition “i” and an amino acid at position “i+4”, wherein i is anyinteger between 12 and 25), by two intervening amino acids, i.e., anamino acid at position “j” and an amino acid at position “j+3,” whereinj is any integer between 12 and 27, or by six intervening amino acids,i.e., an amino acid at position “k” and an amino acid at position “k+7,”wherein k is any integer between 12 and 22. In exemplary embodiments,the bridge or linker is about 8 (or about 7-9) atoms in length and formsbetween side chains of amino acids at positions 12 and 16, or atpositions 16 and 20, or at positions 20 and 24, or at positions 24 and28. The two amino acid side chains can be linked to one another throughnon-covalent bonds, e.g., hydrogen-bonding, ionic interactions, such asthe formation of salt bridges, or by covalent bonds.

In accordance with some embodiments, the Class 3 glucagon relatedpeptide exhibits glucagon/GLP-1 receptor co-agonist activity andcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 11, 47, 48 and 49. In some embodiments, the side chains arecovalently bound to one another, and in some embodiments the two aminoacids are bound to one another to form a lactam ring.

In accordance with some embodiments, the Class 3 glucagon relatedpeptide comprises SEQ ID NO: 45, wherein at least one lactam ring isformed between the side chains of an amino acid pair selected from thegroup consisting of amino acid pairs 12 and 16, 16 and 20, 20 and 24 or24 and 28. In some embodiments, the Class 3 glucagon related peptidecomprises a glucagon peptide analog of SEQ ID NO: 20, wherein thepeptide comprises an intramolecular lactam bridge formed between aminoacid positions 12 and 16 or between amino acid positions 16 and 20. Insome embodiments, the Class 3 glucagon related peptide comprises thesequence of SEQ ID NO: 20, wherein an intramolecular lactam bridge isformed between amino acid positions 12 and 16, between amino acidpositions 16 and 20, or between amino acid positions 20 and 24 and theamino acid at position 29 is glycine, wherein the sequence of SEQ ID NO:29 is linked to the C-terminal amino acid of SEQ ID NO: 20. In a furtherembodiment, the amino acid at position 28 is aspartic acid.

In some specific embodiments, stabilization of the alpha helix structurein the C-terminal portion of the Class 3 glucagon related peptide isachieved through the formation of an intramolecular bridge other than alactam bridge. For example, suitable covalent bonding methods includeany one or more of olefin metathesis, lanthionine-based cyclization,disulfide bridge or modified sulfur-containing bridge formation, the useof α,ω-diaminoalkane tethers, the formation of metal-atom bridges, andother means of peptide cyclization are used to stabilize the alphahelix.

Furthermore, enhanced activity at the GLP-1 receptor may be achieved bystabilizing the alpha-helix structure in the C-terminal portion of theglucagon peptide (around amino acids 12-29) through purposefulintroduction of one or more α,α-disubstituted amino acids at positionsthat retain the desired activity. Such peptides may be considered hereinas a peptide lacking an intramolecular bridge. In some aspects,stabilization of the alpha-helix is accomplished in this manner withoutintroduction of an intramolecular bridge such as a salt bridge orcovalent bond. In some embodiments, one, two, three, four or more ofpositions 16, 17, 18, 19, 20, 21, 24 or 29 of a glucagon peptide issubstituted with an α,α-disubstituted amino acid. For example,substitution of position 16 of the Class 3 glucagon related peptide withamino iso-butyric acid (Aib) enhances GLP-1 activity, in the absence ofa salt bridge or lactam. In some embodiments, one, two, three or more ofpositions 16, 20, 21 or 24 are substituted with Aib.

Enhanced activity at the GLP-1 receptor may be achieved by an amino acidmodification at position 20. In some embodiments, the glutamine atposition 20 is replaced with another hydrophilic amino acid having aside chain that is either charged or has an ability to hydrogen-bond,and is at least about 5 (or about 4-6) atoms in length, for example,lysine, citrulline, arginine, or ornithine.

Increased activity at the GLP-1 receptor is demonstrated in Class 3glucagon related peptides comprising the C-terminal extension of SEQ IDNO: 26. GLP-1 activity in such Class 3 glucagon related peptidescomprising SEQ ID NO: 26 can be further increased by modifying the aminoacid at position 18, 28 or 29, or at position 18 and 29, as describedherein.

A further modest increase in GLP-1 potency may be achieved by modifyingthe amino acid at position 10 to be Trp.

Combinations of the modifications that increase GLP-1 receptor activitymay provide higher GLP-1 activity than any of such modifications takenalone. For example, the Class 3 glucagon related peptides can comprisemodifications at position 16, at position 20, and at the C-terminalcarboxylic acid group, optionally with a covalent bond between the aminoacids at positions 16 and 20; can comprise modifications at position 16and at the C-terminal carboxylic acid group; can comprise modificationsat positions 16 and 20, optionally with a covalent bond between theamino acids at positions 16 and 20; or can comprise modifications atposition 20 and at the C-terminal carboxylic acid group; optionally withthe proviso that the amino acid at position 12 is not Arg; or optionallywith the proviso that the amino acid at position 9 is not Glu.

Modifications Affecting Solubility

Addition of Hydrophilic Moieties

The Class 3 glucagon related peptides can be further modified to improvethe peptide's solubility and stability in aqueous solutions atphysiological pH, while retaining the high biological activity relativeto native glucagon. Hydrophilic moieties as discussed herein can beattached to the Class 3 glucagon related peptide as further discussedherein.

In accordance with some embodiments, introduction of hydrophilic groupsat positions 17, 21, and 24 of the Class 3 glucagon related peptidecomprising SEQ ID NO: 9 or SEQ ID NO: 10 are anticipated to improve thesolubility and stability of the high potency glucagon analog insolutions having a physiological pH. Introduction of such groups alsoincreases duration of action, e.g. as measured by a prolonged half-lifein circulation.

In some embodiments, the Class 3 glucagon related peptide comprises asequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, wherein the side chain of anamino acid residue at one of position 16, 17, 21 or 24 of said Class 3glucagon related peptide further comprises a polyethylene glycol chain,having a molecular weight selected from the range of about 500 to about40,000 Daltons. In some embodiments, the polyethylene glycol chain has amolecular weight selected from the range of about 500 to about 5,000Daltons. In another embodiment, the polyethylene glycol chain has amolecular weight of about 10,000 to about 20,000 Daltons. In yet otherexemplary embodiments the polyethylene glycol chain has a molecularweight of about 20,000 to about 40,000 Daltons.

Suitable hydrophilic moieties include any water soluble polymers knownin the art, including the hydrophilic moieties described herein, homo-or co-polymers of PEG, and a monomethyl-substituted polymer of PEG(mPEG). In accordance with some embodiments the hydrophilic groupcomprises a polyethylene (PEG) chain. More particularly, in someembodiments, the Class 3 glucagon related peptide comprises the sequenceof SEQ ID NO: 6 or SEQ ID NO: 7 wherein a PEG chain is covalently linkedto the side chains of amino acids present at positions 21 and 24 of theClass 3 glucagon related peptide and the carboxy terminal amino acid ofthe Class 3 glucagon related peptide has the carboxylic acid group. Inaccordance with some embodiments, the polyethylene glycol chain has anaverage molecular weight selected from the range of about 500 to about10,000 Daltons.

In accordance with some embodiments, the pegylated Class 3 glucagonrelated peptide comprises two or more polyethylene glycol chainscovalently bound to the Class 3 glucagon related peptide wherein thetotal molecular weight of the glucagon chains is about 1,000 to about5,000 Daltons. In some embodiments the pegylated glucagon agonistcomprises a peptide consisting of SEQ ID NO: 5 or a glucagon agonistanalog of SEQ ID NO: 5, wherein a PEG chain is covalently linked to theamino acid residue at position 21 and at position 24, and wherein thecombined molecular weight of the two PEG chains is about 1,000 to about5,000 Daltons.

Charged C-Terminus

The solubility of the Class 3 glucagon related peptide comprising SEQ IDNO: 20 can be further improved, for example, by introducing one, two,three or more charged amino acid(s) to the C-terminal portion ofglucagon peptide of SEQ ID NO: 20, preferably at a position C-terminalto position 27. Such a charged amino acid can be introduced bysubstituting a native amino acid with a charged amino acid, e.g. atpositions 28 or 29, or alternatively by adding a charged amino acid,e.g. after position 27, 28 or 29. In exemplary embodiments, one, two,three or all of the charged amino acids are negatively charged.Additional modifications, e.g. conservative substitutions, may be madeto the Class 3 glucagon related peptide that still allow it to retainglucagon activity. In some embodiments, an analog of the Class 3glucagon related peptide of SEQ ID NO: 20 is provided wherein the analogdiffers from SEQ ID NO: 20 by 1 to 2 amino acid substitutions atpositions 17-26, and, in some embodiments, the analog differs from thepeptide of SEQ ID NO: 20 by an amino acid substitution at position 20.

Acylation/Alkylation

In accordance with some embodiments, the glucagon peptide is modified tocomprise an acyl or alkyl group, e.g., a C4 to C30 acyl or alkyl group.In some aspects, the acyl group or alkyl group is not naturallyoccurring on an amino acid. In specific aspects, the acyl or alkyl groupis non-native to any naturally-occurring amino acid. Acylation oralkylation can increase the half-life in circulation and/or delay theonset of and/or extend the duration of action and/or improve resistanceto proteases such as DPP-IV. The activity at the glucagon receptor andGLP-1 receptor of the Class 3 glucagon related peptides is maintained,if not substantially enhanced after acylation Further, the potency ofthe acylated analogs were comparable to the unacylated versions of theClass 3 glucagon related peptides, if not substantially enhanced.

In some embodiments, the invention provides a Class 3 glucagon relatedpeptide modified to comprise an acyl group or alkyl group covalentlylinked to the amino acid at position 10 of the glucagon peptide. Theglucagon peptide may further comprise a spacer between the amino acid atposition 10 of the Class 3 glucagon related peptide and the acyl groupor alkyl group. Any of the foregoing Class 3 glucagon related peptidesmay comprise two acyl groups or two alkyl groups, or a combinationthereof.

In a specific aspect of the invention, the acylated Class 3 glucagonrelated peptide comprises the amino acid sequence of any of SEQ ID NOs:534-544 and 546-549.

C-Terminal Truncation

In some embodiments, the Class 3 glucagon related peptides describedherein are further modified by truncation or deletion of one or twoamino acids of the C-terminus of the glucagon peptide (i.e., position 29and/or 28) without affecting activity and/or potency at the glucagon andGLP-1 receptors. In this regard, the Class 3 glucagon related peptidecan comprise amino acids 1-27 or 1-28 of the native glucagon peptide(SEQ ID NO: 1), optionally with one or more modifications describedherein.

In some embodiments, the truncated Class 3 glucagon related peptidecomprises SEQ ID NO: 550 or SEQ ID NO: 551. In another embodiment, thetruncated glucagon agonist peptide comprises SEQ ID NO: 552 or SEQ IDNO: 553.

C-Terminal Extension

In accordance with some embodiments, the Class 3 glucagon relatedpeptides disclosed herein are modified by the addition of a secondpeptide to the carboxy terminus of the glucagon peptide, for example,SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28. In some embodiments, aClass 3 glucagon related peptide having a sequence selected from thegroup consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO:69 is covalently bound through a peptide bond to a second peptide,wherein the second peptide comprises a sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. In afurther embodiment, in Class 3 glucagon related peptides which comprisethe C-terminal extension, the threonine at position 29 of the nativeglucagon peptide is replaced with a glycine. A Class 3 glucagon relatedpeptide having a glycine substitution for threonine at position 29 andcomprising the carboxy terminal extension of SEQ ID NO: 26 is four timesas potent at the GLP-1 receptor as native glucagon modified to comprisethe carboxy terminal extension of SEQ ID NO: 26. Potency at the GLP-1receptor can be further enhanced by an alanine substitution for thenative arginine at position 18.

Accordingly, the Class 3 glucagon related peptide can have a carboxyterminal extension of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28. Inaccordance with some embodiments, Class 3 glucagon related peptidecomprising SEQ ID NO: 33 or SEQ ID NO: 20, further comprises the aminoacid sequence of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked toamino acid 29 of the glucagon peptide. More particularly, the Class 3glucagon related peptide comprises a sequence selected from the groupconsisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14and SEQ ID NO: 15, further comprising the amino acid sequence of SEQ IDNO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked to amino acid 29 of theglucagon peptide. More particularly, the glucagon peptide comprises asequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQID NO: 69, SEQ ID NO: 55 and SEQ ID NO: 56 further comprising the aminoacid sequence of SEQ ID NO: 26 (GPSSGAPPPS) or SEQ ID NO: 29 linked toamino acid 29 of the Class 3 glucagon related peptide. In someembodiments, the Class 3 glucagon related peptide comprises the sequenceof SEQ ID NO: 64.

Other Modifications

Any of the modifications described above with regard to Class 3 glucagonrelated peptides which increase or decrease glucagon receptor activityand which increase GLP-1 receptor activity can be applied individuallyor in combination. Combinations of the modifications that increase GLP-1receptor activity generally provide higher GLP-1 activity than any ofsuch modifications taken alone. Any of the modifications described abovecan also be combined with other modifications described herein inreference to Class 3 glucagon related peptides that confer otherdesirable properties, such as increased solubility and/or stabilityand/or duration of action. Alternatively, any of the modificationsdescribed above can be combined with other modifications describedherein in reference to Class 3 glucagon related peptides that do notsubstantially affect solubility or stability or activity. Exemplarymodifications include but are not limited to:

(A) Improving solubility, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. Such a charged aminoacid can be introduced by substituting a native amino acid with acharged amino acid, e.g. at positions 28 or 29, or alternatively byadding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged. Such modificationsincrease solubility, e.g. provide at least 2-fold, 5-fold, 10-fold,15-fold, 25-fold, 30-fold or greater solubility relative to nativeglucagon at a given pH between about 5.5 and 8, e.g., pH 7, whenmeasured after 24 hours at 25° C.

(B) Increasing solubility and duration of action or half-life incirculation by addition of a hydrophilic moiety such as a polyethyleneglycol chain, as described herein, e.g. at position 16, 17, 20, 21, 24or 29, or at the C-terminal amino acid of the peptide.

(C) Increasing stability by modification of the aspartic acid atposition 15, for example, by deletion or substitution with glutamicacid, homoglutamic acid, cysteic acid or homocysteic acid. Suchmodifications can reduce degradation or cleavage at a pH within therange of 5.5 to 8, especially in acidic or alkaline buffers, forexample, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% ofthe original peptide after 24 hours at 25° C.

(D) Increasing stability by modification of the methionine at position27, for example, by substitution with leucine or norleucine. Suchmodifications can reduce oxidative degradation. Stability can also beincreased by modification of the Gln at position 20 or 24, e.g. bysubstitution with Ser, Thr, Ala or Aib. Such modifications can reducedegradation that occurs through deamidation of Gln. Stability can beincreased by modification of Asp at position 21, e.g. by substitutionwith Glu. Such modifications can reduce degradation that occurs throughdehydration of Asp to form a cyclic succinimide intermediate followed byisomerization to iso-aspartate.

(E) Increasing resistance to dipeptidyl peptidase IV (DPP IV) cleavageby modification of the amino acid at position 1 or 2 with the DPP-IVresistant amino acids described herein and including modification of theamino acid at position 2 with N-methyl-alanine.

(F) Conservative or non-conservative substitutions, additions ordeletions that do not affect activity, for example, conservativesubstitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14,16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; deletions at one or more ofpositions 27, 28 or 29; or a deletion of amino acid 29 optionallycombined with a C-terminal amide or ester in place of the C-terminalcarboxylic acid group;

(G) Adding C-terminal extensions as described herein;

(H) Increasing half-life in circulation and/or extending the duration ofaction and/or delaying the onset of action, for example, throughacylation or alkylation of the glucagon peptide, as described herein;

(I) Homodimerization or heterodimerization as described herein.

Other modifications include substitution of His at position 1 with alarge, aromatic amino acid (e.g., Tyr, Phe, Trp or amino-Phe); Ser atposition 2 with Ala; substitution of Tyr at position 10 with Val or Phe;substitution of Lys at position 12 with Arg; substitution of Asp atposition 15 with Glu; substitution of Ser at position 16 with Thr orAib.

Class 3 glucagon related peptides with GLP-1 activity that contain anon-conservative substitution of His at position 1 with a large,aromatic amino acid (e.g., Tyr) can retain GLP-1 activity provided thatthe alpha-helix is stabilized via an intramolecular bridge, e.g., suchas any of those described herein.

Conjugates and Fusions

The Class 3 glucagon related peptide can be linked, optionally viacovalent bonding and optionally via a linker, to a conjugate moiety.

The Class 3 glucagon related peptide also can be part of a fusionpeptide or protein wherein a second peptide or polypeptide has beenfused to a terminus, e.g., the carboxy terminus of the Class 3 glucagonrelated peptide.

More particularly, the fusion Class 3 glucagon related peptide maycomprise a glucagon agonist of SEQ ID NO: 55, SEQ ID NO: 9 or SEQ ID NO:10 further comprising an amino acid sequence of SEQ ID NO: 26(GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked toamino acid 29 of the glucagon peptide. In some embodiments, the aminoacid sequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) orSEQ ID NO: 28 (KRNR) is bound to amino acid 29 of the Class 3 glucagonrelated peptide through a peptide bond. Applicants have discovered thatin Class 3 glucagon related peptide fusion peptides comprising theC-terminal extension peptide of Exendin-4 (e.g., SEQ ID NO: 26 or SEQ IDNO: 29), substitution of the native threonine residue at position 29with glycine dramatically increases GLP-1 receptor activity. This aminoacid substitution can be used in conjunction with other modificationsdisclosed herein with regard to Class 3 glucagon related peptides toenhance the affinity of the glucagon analogs for the GLP-1 receptor. Forexample, the T29G substitution can be combined with the S16E and N20Kamino acid substitutions, optionally with a lactam bridge between aminoacids 16 and 20, and optionally with addition of a PEG chain asdescribed herein. In some embodiments, a Class 3 glucagon relatedpeptide comprises the sequence of SEQ ID NO: 64. In some embodiments,the Class 3 glucagon related peptide portion of the glucagon fusionpeptide is selected from the group consisting of SEQ ID NO: 55, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 wherein a PEG chain,when present at positions 17, 21, 24, or the C-terminal amino acid, orat both 21 and 24, is selected from the range of 500 to 40,000 Daltons.More particularly, in some embodiments, the Class 3 glucagon relatedpeptide segment is selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 8, and SEQ ID NO: 63, wherein the PEG chain is selected fromthe range of 500 to 5,000. In some embodiments, the Class 3 glucagonrelated peptide is a fusion peptide comprising the sequence of SEQ IDNO: 55 and SEQ ID NO: 65 wherein the peptide of SEQ ID NO: 65 is linkedto the carboxy terminus of SEQ ID NO: 55.

In accordance with some embodiments, an additional chemical modificationof the Class 3 glucagon related peptide of SEQ ID NO: 10 bestowsincreased GLP-1 receptor potency to a point where the relative activityat the glucagon and GLP-1 receptors is virtually equivalent.Accordingly, in some embodiments, a Class 3 glucagon related peptidecomprises a terminal amino acid comprising an amide group in place ofthe carboxylic acid group that is present on the native amino acid. Therelative activity of the Class 3 glucagon related peptide at therespective glucagon and GLP-1 receptors can be adjusted by furthermodifications to the Class 3 glucagon related peptide to produce analogsdemonstrating about 40% to about 500% or more of the activity of nativeglucagon at the glucagon receptor and about 20% to about 200% or more ofthe activity of native GLP-1 at the GLP-1 receptor, e.g. 50-fold,100-fold or more increase relative to the normal activity of glucagon atthe GLP-1 receptor. In some embodiments, the glucagon peptides describedherein exhibit up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000%of the activity of native glucagon at the glucagon receptor. In someembodiments, the glucagon peptides described herein exhibit up to about100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of nativeGLP-1 at the GLP-1 receptor.

Exemplary Embodiments

In accordance with some embodiments, a glucagon analog is providedcomprising the sequence of SEQ ID NO: 55, wherein said analog differsfrom SEQ ID NO: 55 by 1 to 3 amino acids, selected from positions 1, 2,3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 24, 27, 28, and 29, whereinsaid glucagon peptide exhibits at least 20% of the activity of nativeGLP-1 at the GLP-1 receptor.

In accordance with some embodiments a glucagon/GLP-1 receptor co-agonistis provided comprising the sequence:

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 33) wherein the Xaa at position 15 is selected from thegroup of amino acids consisting of Asp, Glu, cysteic acid, homoglutamicacid and homocysteic acid, Xaa at position 16 is selected from the groupof amino acids consisting of Ser, Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaa at position 20 is Gln or Lys, the Xaa atposition 24 is Gln or Glu, the Xaa at position 28 is Asn, Lys or anacidic amino acid, the Xaa at position 29 is Thr, Gly or an acidic aminoacid, and R is COOH or CONH₂, with the proviso that when position 16 isserine, position 20 is Lys, or alternatively when position 16 is serinethe position 24 is Glu and either position 20 or position 28 is Lys. Insome embodiments the glucagon/GLP-1 receptor co-agonist comprises thesequence of SEQ ID NO: 33 wherein the amino acid at position 28 isaspartic acid and the amino acid at position 29 is glutamic acid. Inanother embodiment the amino acid at position 28 is the nativeasparagine, the amino acid at position 29 is glycine and the amino acidsequence of SEQ ID NO: 29 or SEQ ID NO: 65 is covalently linked to thecarboxy terminus of SEQ ID NO: 33.

In some embodiments a co-agonist is provided comprising the sequence ofSEQ ID NO: 33 wherein an additional acidic amino acid added to thecarboxy terminus of the peptide. In a further embodiment the carboxyterminal amino acid of the glucagon analog has an amide in place of thecarboxylic acid group of the natural amino acid. In some embodiments theglucagon analog comprises a sequence selected from the group consistingof SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ IDNO: 44.

In accordance with some embodiments a glucagon peptide analog of SEQ IDNO: 33 is provided, wherein said analog differs from SEQ ID NO: 33 by 1to 3 amino acids, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14,17, 18, 19, 21 and 27, with the proviso that when the amino acid atposition 16 is serine, either position 20 is lysine, or a lactam bridgeis formed between the amino acid at position 24 and either the aminoacid at position 20 or position 28. In accordance with some embodimentsthe analog differs from SEQ ID NO: 33 by 1 to 3 amino acids selectedfrom positions 1, 2, 3, 21 and 27. In some embodiments the glucagonpeptide analog of SEQ ID NO: 33 differs from that sequence by 1 to 2amino acids, or in some embodiments by a single amino acid, selectedform positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27,with the proviso that when the amino acid at position 16 is serine,either position 20 is lysine, or a lactam bridge is formed between theamino acid at position 24 and either the amino acid at position 20 orposition 28.

In accordance with another embodiment a relatively selective GLP-1receptor agonist is provided comprising the sequenceNH₂-His-Ser-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 53) wherein the Xaa at position 3 is selected from the groupof amino acids consisting of Glu, Orn or Nle, the Xaa at position 15 isselected from the group of amino acids consisting of Asp, Glu, cysteicacid, homoglutamic acid and homocysteic acid, Xaa at position 16 isselected from the group of amino acids consisting of Ser, Glu, Gln,homoglutamic acid and homocysteic acid, the Xaa at position 20 is Gln orLys, the Xaa at position 24 is Gln or Glu, the Xaa at position 28 isAsn, Lys or an acidic amino acid, the Xaa at position 29 is Thr, Gly oran acidic amino acid, and R is COOH, CONH2, SEQ ID NO: 26 or SEQ ID NO:29, with the proviso that when position 16 is serine, position 20 isLys, or alternatively when position 16 is serine the position 24 is Gluand either position 20 or position 28 is Lys. In some embodiments theamino acid at position 3 is glutamic acid. In some embodiments theacidic amino acid substituted at position 28 and/or 29 is aspartic acidor glutamic acid. In some embodiments the glucagon peptide, including aco-agonist peptide, comprises the sequence of SEQ ID NO: 33 furthercomprising an additional acidic amino acid added to the carboxy terminusof the peptide. In a further embodiment the carboxy terminal amino acidof the glucagon analog has an amide in place of the carboxylic acidgroup of the natural amino acid.

In accordance with some embodiments a glucagon/GLP-1 receptor co-agonistis provided comprising a modified glucagon peptide selected from thegroup consisting of:

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 34), wherein the Xaa at position 15 is selected from thegroup of amino acids consisting of Asp, Glu, cysteic acid, homoglutamicacid and homocysteic acid, Xaa at position 16 is selected from the groupof amino acids consisting of Ser, Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaa at position 20 is Gln or Lys, the Xaa atposition 24 is Gln or Glu and the Xaa at position 28 is Asn, Asp or Lys,R is COOH or CONH₂, the Xaa at position 29 is Thr or Gly, and R is COOH,CONH₂, SEQ ID NO: 26 or SEQ ID NO: 29, with the proviso that whenposition 16 is serine, position 20 is Lys, or alternatively whenposition 16 is serine the position 24 is Glu and either position 20 orposition 28 is Lys. In some embodiments R is CONH₂, the Xaa at position15 is Asp, the Xaa at position 16 is selected from the group of aminoacids consisting of Glu, Gln, homoglutamic acid and homocysteic acid,the Xaas at positions 20 and 24 are each Gln the Xaa at position 28 isAsn or Asp and the Xaa at position 29 is Thr. In some embodiments theXaas at positions 15 and 16 are each Glu, the Xaas at positions 20 and24 are each Gln, the Xaa at position 28 is Asn or Asp, the Xaa atposition 29 is Thr and R is CONH₂.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, applicants anticipate that one or moreof the amino acids located at positions at positions 2, 5, 7, 10, 11,12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide of SEQID NO: 11 can be substituted with an amino acid different from thatpresent in the native glucagon peptide, and still retain activity at theglucagon receptor. In some embodiments the methionine residue present atposition 27 of the native peptide is changed to leucine or norleucine toprevent oxidative degradation of the peptide. In another embodiment theamino acid at position 20 is substituted with Lys, Arg, Orn orCitrullene and/or position 21 is substituted with Glu, homoglutamic acidor homocysteic acid.

In some embodiments a glucagon analog of SEQ ID NO: 20 is providedwherein 1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11,13, 14, 17, 18, 19, 21, 27, 28 or 29 of the analog differ from thecorresponding amino acid of SEQ ID NO: 1, with the proviso that when theamino acid at position 16 is serine, position 20 is Lys, oralternatively when position 16 is serine the position 24 is Glu andeither position 20 or position 28 is Lys. In accordance with anotherembodiment a glucagon analog of SEQ ID NO: 20 is provided wherein 1 to 3amino acids selected from positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18,19, 20, 21, 27, 28 or 29 of the analog differ from the correspondingamino acid of SEQ ID NO: 1. In another embodiment, a glucagon analog ofSEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 11 is provided wherein 1 to 2amino acids selected from positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18,19, 20 or 21 of the analog differ from the corresponding amino acid ofSEQ ID NO: 1, and in a further embodiment the one to two differing aminoacids represent conservative amino acid substitutions relative to theamino acid present in the native glucagon sequence (SEQ ID NO: 1). Insome embodiments a glucagon peptide of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14 or SEQ ID NO: 15 is provided wherein the glucagon peptidefurther comprises one, two or three amino acid substitutions atpositions selected from positions 2, 5, 7, 10, 11, 13, 14, 17, 18, 19,20, 21, 27 or 29. In some embodiments the substitutions at positions 2,5, 7, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 27 or 29 are conservativeamino acid substitutions.

In accordance with some embodiments a glucagon/GLP-1 receptor co-agonistis provided comprising a variant of the sequence of SEQ ID NO 33,wherein 1 to 10 amino acids selected from positions 16, 17, 18, 20, 21,23, 24, 27, 28 and 29, respectively, of the variant differ from thecorresponding amino acid of SEQ ID NO: 1. In accordance with someembodiments a variant of the sequence of SEQ ID NO 33 is providedwherein the variant differs from SEQ ID NO: 33 by one or more amino acidsubstitutions selected from the group consisting of Gln17, Ala18, Glu21,Ile23, Ala24, Val27 and Gly29. In accordance with some embodiments aglucagon/GLP-1 receptor co-agonist is provided comprising variants ofthe sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected frompositions 17-26 of the variant differ from the corresponding amino acidof SEQ ID NO: 1. In accordance with some embodiments a variant of thesequence of SEQ ID NO 33 is provided wherein the variant differs fromSEQ ID NO: 33 by an amino acid substitution selected from the groupconsisting of Gln17, Ala18, Glu21, Ile23 and Ala24. In accordance withsome embodiments a variant of the sequence of SEQ ID NO 33 is providedwherein the variant differs from SEQ ID NO: 33 by an amino acidsubstitution at position 18 wherein the substituted amino acid isselected from the group consisting of Ala, Ser, Thr, and Gly. Inaccordance with some embodiments a variant of the sequence of SEQ ID NO33 is provided wherein the variant differs from SEQ ID NO: 33 by anamino acid substitution of Ala at position 18. Such variations areencompassed by SEQ ID NO: 55. In another embodiment a glucagon/GLP-1receptor co-agonist is provided comprising variants of the sequence ofSEQ ID NO 33, wherein 1 to 2 amino acids selected from positions 17-22of the variant differ from the corresponding amino acid of SEQ ID NO: 1,and in a further embodiment a variant of SEQ ID NO 33 is providedwherein the variant differs from SEQ ID NO: 33 by for 2 amino acidsubstitutions at positions 20 and 21. In accordance with someembodiments a glucagon/GLP-1 receptor co-agonist is provided comprisingthe sequence:

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Xaa-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 51), wherein the Xaa at position 15 is Asp, Glu, cysteicacid, homoglutamic acid or homocysteic acid, the Xaa at position 16 isSer, Glu, Gln, homoglutamic acid or homocysteic acid, the Xaa atposition 20 is Gln, Lys, Arg, Orn or citrulline, the Xaa at position 21is Asp, Glu, homoglutamic acid or homocysteic acid, the Xaa at position24 is Gln or Glu, the Xaa at position 28 is Asn, Lys or an acidic aminoacid, the Xaa at position 29 is Thr or an acid amino acid and R is COOHor CONH2. In some embodiments R is CONH2. In accordance with someembodiments a glucagon/GLP-1 receptor co-agonist is provided comprisinga variant of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, whereinthe variant differs from said sequence by an amino acid substitution atposition 20. In some embodiments the amino acid substitution is selectedform the group consisting of Lys, Arg, Orn or citrulline for position20.

In some embodiments a glucagon agonist is provided comprising an analogpeptide of SEQ ID NO: 34 wherein the analog differs from SEQ ID NO: 34by having an amino acid other than serine at position 2. In someembodiments the serine residue is substituted with aminoisobutyric acid,D-alanine, and in some embodiments the serine residue is substitutedwith aminoisobutyric acid. Such modifications suppresses cleavage bydipeptidyl peptidase IV while retaining the inherent potency of theparent compound (e.g. at least 75, 80, 85, 90, 95% or more of thepotency of the parent compound). In some embodiments the solubility ofthe analog is increased, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. In exemplaryembodiments, one, two, three or all of the charged amino acids arenegatively charged. In another embodiment the analog further comprisesan acidic amino acid substituted for the native amino acid at position28 or 29 or an acidic amino acid added to the carboxy terminus of thepeptide of SEQ ID NO: 34.

In some embodiments the glucagon analogs disclosed herein are furthermodified at position 1 or 2 to reduce susceptibility to cleavage bydipeptidyl peptidase IV. In some embodiments a glucagon analog of SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQID NO: 15 is provided wherein the analog differs from the parentmolecule by a substitution at position 2 and exhibits reducedsusceptibility (i.e., resistance) to cleavage by dipeptidyl peptidaseIV. More particularly, in some embodiments position 2 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of D-serine, D-alanine, valine, amino n-butyric acid,glycine, N-methyl serine and aminoisobutyric acid. In some embodimentsposition 2 of the analog peptide is substituted with an amino acidselected from the group consisting of D-serine, D-alanine, glycine,N-methyl serine and aminoisobutyric acid. In another embodiment position2 of the analog peptide is substituted with an amino acid selected fromthe group consisting of D-serine, glycine, N-methyl serine andaminoisobutyric acid. In some embodiments the amino acid at position 2is not D-serine. In some embodiments the glucagon peptide comprises thesequence of SEQ ID NO: 21 or SEQ ID NO: 22.

In some embodiments a glucagon analog of SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is providedwherein the analog differs from the parent molecule by a substitution atposition 1 and exhibits reduced susceptibility (i.e., resistance) tocleavage by dipeptidyl peptidase IV. More particularly, position 1 ofthe analog peptide is substituted with an amino acid selected from thegroup consisting of D-histidine, alpha, alpha-dimethyl imidiazole aceticacid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazoleacetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. In another embodiment a glucagon agonist is providedcomprising an analog peptide of SEQ ID NO: 34 wherein the analog differsfrom SEQ ID NO: 34 by having an amino acid other than histidine atposition 1. In some embodiments the solubility of the analog isincreased, for example, by introducing one, two, three or more chargedamino acid(s) to the C-terminal portion of native glucagon, preferablyat a position C-terminal to position 27. In exemplary embodiments, one,two, three or all of the charged amino acids are negatively charged. Inanother embodiment the analog further comprises an acidic amino acidsubstituted for the native amino acid at position 28 or 29 or an acidicamino acid added to the carboxy terminus of the peptide of SEQ ID NO:34. In some embodiments the acidic amino acid is aspartic acid orglutamic acid.

In some embodiments the glucagon/GLP-1 receptor co-agonist comprises asequence of SEQ ID NO: 20 further comprising an additional carboxyterminal extension of one amino acid or a peptide selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. Inthe embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in some embodiments the additionalcarboxy terminus amino acid has an amide group in place of thecarboxylic acid of the native amino acid. In some embodiments theadditional amino acid is selected from the group consisting of glutamicacid, aspartic acid and glycine.

In an alternative embodiment a glucagon/GLP-1 receptor co-agonist isprovided wherein the peptide comprises at least one lactam ring formedbetween the side chain of a glutamic acid residue and a lysine residue,wherein the glutamic acid residue and a lysine residue are separated bythree amino acids. In some embodiments the carboxy terminal amino acidof the lactam bearing glucagon peptide has an amide group in place ofthe carboxylic acid of the native amino acid. More particularly, in someembodiments a glucagon and GLP-1 co-agonist is provided comprising amodified glucagon peptide selected from the group consisting of:

(SEQ ID NO: 66) NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 67)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 68)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 69)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Xaa-R (SEQ ID NO: 16)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Asn-Thr-R (SEQ ID NO: 17)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R (SEQ ID NO: 18)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R

wherein Xaa at position 28 is Asp, or Asn, the Xaa at position 29 is Thror Gly, R is selected from the group consisting of COOH, CONH₂, glutamicacid, aspartic acid, glycine, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ IDNO: 28, and a lactam bridge is formed between Lys at position 12 and Gluat position 16 for SEQ ID NO: 66, between Glu at position 16 and Lys atposition 20 for SEQ ID NO: 67, between Lys at position 20 and Glu atposition 24 for SEQ ID NO: 68, between Glu at position 24 and Lys atposition 28 for SEQ ID NO: 69, between Lys at position 12 and Glu atposition 16 and between Lys at position 20 and Glu at position 24 forSEQ ID NO: 16, between Lys at position 12 and Glu at position 16 andbetween Glu at position 24 and Lys at position 28 for SEQ ID NO: 17 andbetween Glu at position 16 and Lys at position 20 and between Glu atposition 24 and Lys at position 28 for SEQ ID NO: 18. In someembodiments R is selected from the group consisting of COOH, CONH₂,glutamic acid, aspartic acid, glycine, the amino acid at position 28 isAsn, and the amino acid at position 29 is threonine. In some embodimentsR is CONH₂, the amino acid at position 28 is Asn and the amino acid atposition 29 is threonine. In another embodiment R is selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 65 andthe amino acid at position 29 is glycine.

In a further embodiment the glucagon/GLP-1 receptor co-agonist isselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17and SEQ ID NO: 18, wherein the peptide further comprises an additionalcarboxy terminal extension of one amino acid or a peptide selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.In some embodiments the terminal extension comprises the sequence of SEQID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 and the glucagon peptidecomprises the sequence of SEQ ID NO: 55. In some embodiments theglucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO:33 wherein the amino acid at position 16 is glutamic acid, the aminoacid at position 20 is lysine, the amino acid at position 28 isasparagine and the amino acid sequence of SEQ ID No: 26 or SEQ ID NO: 29is linked to the carboxy terminus of SEQ ID NO: 33.

In the embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in some embodiments the amino acid hasan amide group in place of the carboxylic acid of the native amino acid.In some embodiments the additional amino acid is selected from the groupconsisting of glutamic acid and aspartic acid and glycine. In theembodiments wherein the glucagon agonist analog further comprises acarboxy terminal extension, the carboxy terminal amino acid of theextension, in some embodiments, ends in an amide group or an ester grouprather than a carboxylic acid.

In another embodiment the glucagon/GLP-1 receptor co-agonist comprisesthe sequence:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Xaa-CONH₂(SEQ ID NO: 19), wherein the Xaa at position 30 represents any aminoacid. In some embodiments Xaa is selected from one of the 20 commonamino acids, and in some embodiments the amino acid is glutamic acid,aspartic acid or glycine. The solubility of this peptide can be furtherimproved by covalently linking a PEG chain to the side chain of aminoacid at position 17, 21, 24 or 30 of SEQ ID NO: 19. In a furtherembodiment the peptide comprises an additional carboxy terminalextension of a peptide selected from the group consisting of SEQ ID NO:26, SEQ ID NO: 27 and SEQ ID NO: 28. In accordance with some embodimentsthe glucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ IDNO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.

Additional site specific modifications internal to the glucagon sequenceof SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 andSEQ ID NO: 64 can be made to yield a set of glucagon agonists thatpossess variable degrees of GLP-1 agonism. Accordingly, peptides thatpossess virtually identical in vitro potency at each receptor have beenprepared and characterized. Similarly, peptides with tenfold selectivelyenhanced potency at each of the two receptors have been identified andcharacterized. As noted above substitution of the serine residue atposition 16 with glutamic acid enhances the potency of native glucagonat both the Glucagon and GLP-1 receptors, but maintains approximately atenfold selectivity for the glucagon receptor. In addition bysubstituting the native glutamine at position 3 with glutamic acid (SEQID NO: 22) generates a glucagon analog that exhibits approximately atenfold selectivity for the GLP-1 receptor.

The solubility of the glucagon/GLP-1 co-agonist peptides can be furtherenhanced in aqueous solutions at physiological pH, while retaining thehigh biological activity relative to native glucagon by the introductionof hydrophilic groups at positions 16, 17, 21, and 24 of the peptide, orby the addition of a single modified amino acid (i.e., an amino acidmodified to comprise a hydrophilic group) at the carboxy terminus of theglucagon/GLP-1 co-agonist peptide. In accordance with some embodimentsthe hydrophilic group comprises a polyethylene (PEG) chain. Moreparticularly, in some embodiments the glucagon peptide comprises thesequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18 wherein a PEG chain is covalently linked to the side chain of anamino acids at position 16, 17, 21, 24, 29 or the C-terminal amino acidof the glucagon peptide, with the proviso that when the peptidecomprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13the polyethylene glycol chain is covalently bound to an amino acidresidue at position 17, 21 or 24, when the peptide comprises SEQ ID NO:14 or SEQ ID NO: 15 the polyethylene glycol chain is covalently bound toan amino acid residue at position 16, 17 or 21, and when the peptidecomprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 the polyethyleneglycol chain is covalently bound to an amino acid residue at position 17or 21.

In some embodiments the glucagon peptide comprises the sequence of SEQID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 17,21, 24, or the C-terminal amino acid of the glucagon peptide, and thecarboxy terminal amino acid of the peptide has an amide group in placeof the carboxylic acid group of the native amino acid. In someembodiments the glucagon/GLP-1 receptor co-agonist peptide comprises asequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18 and SEQ ID NO: 19, wherein a PEG chain is covalently linked tothe side chain of an amino acid at position 17, 21 or 24 of SEQ ID NO:12, SEQ ID NO: 13 and SEQ ID NO: 19, or at position 16, 17 or 21 of SEQID NO: 14 and SEQ ID NO: 15 or at position 17 or 21 of SEQ ID NO: 16,SEQ ID NO: 17 and SEQ ID NO: 18 of the glucagon peptide. In anotherembodiment the glucagon/GLP-1 receptor co-agonist peptide comprises thesequence of SEQ ID NO: 11 or SEQ ID NO: 19, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 17, 21or 24 or the C-terminal amino acid of the glucagon peptide.

In accordance with some embodiments, and subject to the provisolimitations described in the preceding paragraphs, the glucagonco-agonist peptide is modified to contain one or more amino acidsubstitution at positions 16, 17, 21, 24, or 29 or the C-terminal aminoacid, wherein the native amino acid is substituted with an amino acidhaving a side chain suitable for crosslinking with hydrophilic moieties,including for example, PEG. The native peptide can be substituted with anaturally occurring amino acid or a synthetic (non-naturally occurring)amino acid. Synthetic or non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.Alternatively, the amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG, canbe added to the carboxy terminus of any of the glucagon analogsdisclosed herein. In accordance with some embodiments an amino acidsubstitution is made in the glucagon/GLP-1 receptor co-agonist peptideat a position selected from the group consisting of 16, 17, 21, 24, or29 replacing the native amino acid with an amino acid selected from thegroup consisting of lysine, cysteine, ornithine, homocysteine and acetylphenylalanine, wherein the substituting amino acid further comprises aPEG chain covalently bound to the side chain of the amino acid. In someembodiments a glucagon peptide selected form the group consisting of SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 is further modified tocomprise a PEG chain is covalently linked to the side chain of an aminoacid at position 17 or 21 of the glucagon peptide. In some embodimentsthe pegylated glucagon/GLP-1 receptor co-agonist further comprises thesequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55 or SEQ ID NO: 56, further comprising a C-terminal extension ofSEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 linked to the C-terminalamino acid of SEQ ID NO: 55 or SEQ ID NO: 56, and optionally furthercomprising a PEG chain covalently linked to the side chain of an aminoacids at position 17, 18, 21, 24 or 29 or the C-terminal amino acid ofthe peptide. In another embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 55 or SEQ ID NO: 56, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 21 or24 of the glucagon peptide and the peptide further comprises aC-terminal extension of SEQ ID NO: 26, or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55, or SEQ ID NO: 33 or SEQ ID NO: 34, wherein an additionalamino acid is added to the carboxy terminus of SEQ ID NO: 33 or SEQ IDNO: 34, and a PEG chain is covalently linked to the side chain of theadded amino acid. In a further embodiment, the pegylated glucagon analogfurther comprises a C-terminal extension of SEQ ID NO: 26 or SEQ ID NO:29 linked to the C-terminal amino acid of SEQ ID NO: 33 or SEQ ID NO:34. In another embodiment the glucagon peptide comprises the sequence ofSEQ ID NO: 19, wherein a PEG chain is covalently linked to the sidechain of the amino acid at position 30 of the glucagon peptide and thepeptide further comprises a C-terminal extension of SEQ ID NO: 26 or SEQID NO: 29 linked to the C-terminal amino acid of SEQ ID NO: 19.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with some embodiments the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 10,000 Daltons. In some embodiments the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 1,000 to about 5,000 Daltons. In an alternative embodiment thepolyethylene glycol chain has an average molecular weight selected fromthe range of about 10,000 to about 20,000 Daltons. In accordance withsome embodiments the pegylated glucagon peptide comprises two or morepolyethylene glycol chains covalently bound to the glucagon peptidewherein the total molecular weight of the glucagon chains is about 1,000to about 5,000 Daltons. In some embodiments the pegylated glucagonagonist comprises a peptide consisting of SEQ ID NO: 5 or a glucagonagonist analog of SEQ ID NO: 5, wherein a PEG chain is covalently linkedto the amino acid residue at position 21 and at position 24, and whereinthe combined molecular weight of the two PEG chains is about 1,000 toabout 5,000 Daltons.

In certain exemplary embodiments, the glucagon peptide comprises theamino acid sequence of SEQ ID NO: 1 with up to ten amino acidmodifications and comprises an amino acid at position 10 which isacylated or alkylated. In some embodiments, the amino acid at position10 is acylated or alkylated with a C4 to C30 fatty acid. In certainaspects, the amino acid at position 10 comprises an acyl group or analkyl group which is non-native to a naturally-occurring amino acid.

In certain embodiments, the glucagon peptide comprising an amino acid atposition 10 which is acylated or alkylated comprises a stabilized alphahelix. Accordingly, in certain aspects, the glucagon peptide comprisesan acyl or alkyl group as described herein and an intramolecular bridge,e.g., a covalent intramolecular bridge (e.g., a lactam bridge) betweenthe side chains of an amino acid at position i and an amino acid atposition i+4, wherein i is 12, 16, 20, or 24. Alternatively oradditionally, the glucagon peptide comprises an acyl or alkyl group asdescribed herein and one, two, three or more of positions 16, 20, 21and/or 24 of the glucagon peptide are substituted with anα,α-disubstituted amino acid, e.g., Aib. In some instances, thenon-native glucagon peptide comprises Glu at position 16 and Lys atposition 20, wherein optionally a lactam bridge lnkes the Glu and theLys, and, optionally, the glucagon peptide further comprises one or moremodifications selected from the group consisting of: Gln at position 17,Ala at position 18, Glu at position 21, Ile at position 23, and Ala atposition 24.

Also, in any of the embodiments, wherein the glucagon peptide comprisesan amino acid at position 10 which is acylated or alkylated, theglucagon peptide can further comprise a C-terminal amide in lieu of theC-terminal alpha carboxylate.

In some embodiments, the glucagon peptide comprising an acyl or alkylgroup as described herein further comprises an amino acid substitutionat position 1, at position 2, or at positions 1 and 2, wherein the aminoacid substitution(s) achieve DPP-IV protease resistance. For example,the His at position 1 may be substituted with an amino acid selectedfrom the group consisting of: D-histidine, alpha, alpha-dimethylimidiazole acetic acid (DMIA), N-methyl histidine, alpha-methylhistidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine,acetyl-histidine and homo-histidine. Alternatively or additionally, theSer at position 2 may be substituted with an amino acid selected fromthe group consisting of: D-serine, alanine, D-alanine, valine, glycine,N-methyl serine, N-methyl alanine, and amino isobutyric acid. In someembodiments the amino acid at position 2 is not D-serine.

The glucagon peptide comprising the amino acid at position 10 which isacylated or alkylated as described herein can comprise any amino acidsequence which is substantially related to SEQ ID NO: 1. For instance,the glucagon peptide comprises SEQ ID NO: 1 with up to 10 amino acidmodifications (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 modifications). Incertain embodiments, the amino acid sequence of the acylated oralkylated glucagon peptide is greater than 25% identical to SEQ ID NO: 1(e.g., greater than 30%, 35%, 40%, 50%, 60%, 70% 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or nearly 100% identical to SEQ ID NO: 1). Incertain specific embodiments, the glucagon peptide is one whichcomprises SEQ ID NOs: 55 with an amino acid at position 10 acylated oralkylated as described herein. The glucagon peptide can be any of SEQ IDNOs: 55, 55 with 1 or 2 amino acid modifications, 2-4, 9-18, 20, 23-25,33, 40-44, 53, 56, 61, 62, 64, 66-514, and 534.

The acyl or alkyl group of these embodiments may be any acyl or alkylgroup described herein. For example, the acyl group may be a C4 to C30(e.g., C8 to C24) fatty acyl group and the alkyl group may be a C4 toC30 (e.g., C8 to C24) alkyl group.

The amino acid to which the acyl or alkyl group is attached may be anyof the amino acids described herein, e.g., an amino acid of any ofFormula I (e.g., Lys), Formula II, and Formula III.

In some embodiments, the acyl group or alkyl group is directly attachedto the amino acid at position 10. In some embodiments, the acyl or alkylgroup is attached to the amino acid at position 10 via a spacer, suchas, for example, a spacer which is 3 to 10 atoms in length, e.g., anamino acid or dipeptide. Suitable spacers for purposes of attaching anacyl or alkyl group are described herein.

In accordance with some embodiments, the Class 3 glucagon relatedpeptide may be an analog of any of the foregoing Class 3 glucagonrelated peptides as described herein, which analog exhibits agonistactivity at the GIP receptor. The activity level of the analog at theglucagon receptor, the GLP-1 receptor, and the GIP receptor, the potencyat each of these receptors, and the selectivity for each of thesereceptors may be in accordance with the teachings of Class 2 glucagonrelated peptides described herein. See, the teachings under thesubsection of the Class 2 glucagon related peptide section entitled“Activity.”

In some embodiments of the invention, an analog of a glucagon peptide,which analog exhibits agonist activity at the GIP receptor, is provided.The analog in certain embodiments comprises the amino acid sequence ofSEQ ID NO: 1 with at least one amino acid modification (optionally, upto 15 amino acid modifications), and an extension of 1 to 21 amino acidsC-terminal to the amino acid at position 29 of the analog.

In certain aspects, the analogs comprise at least one amino acidmodification and up to 15 amino acid modifications (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid modifications, up to 10amino acid modifications). In certain embodiments, the analogs compriseat least one amino acid modification at up to 10 amino acidmodifications and additional conservative amino acid modifications.Conservative amino acid modifications are described herein.

In some aspects, at least one of the amino acid modifications confers astabilized alpha helix structure in the C-terminal portion of theanalog. Modifications which achieve a stabilized alpha helix structureare described herein. See, for example, the teachings under the sectionentitled Stabilization of the alpha helix/Intramolecular bridges. Insome aspects, the analog comprises an intramolecular bridge (e.g., acovalent intramolecular bridge, a non-covalent intramolecular bridge)between the side chains of two amino acids of the analog. In certainaspects, an intramolecular bridge links the side chains of the aminoacids at positions i and i+4, wherein i is 12, 13, 16, 17, 20, or 24. Inother aspects, an intramolecular bridge connects the side chains of theamino acids at positions j and j+3, wherein j is 17, or at positions kand k+7″ wherein k is any integer between 12 and 22. In certainembodiments, the intramolecular bridge is a covalent intramolecularbridge, e.g., a lactam bridge. In specific aspects, the lactam bridgeconnects the side chains of the amino acids at positions 16 and 20. Inparticular aspects, one of the amino acids at positions 16 and 20 is apositive-charged amino acid and the other is a negative-charged aminoacid. For example, the analog can comprise a lactam bridge connectingthe side chains of a Glu at position 16 and a Lys at position 20. Inother aspects, the negative-charged amino acid and the positive-chargedamino acid form a salt bridge. In this instance, the intramolecularbridge is a non-covalent intramolecular bridge.

In particular aspects, the amino acid modification which confers astabilized alpha helix is an insertion or substitution of an amino acidof SEQ ID NO: 1 with an α,α-disubstituted amino acid. Suitableα,α-disubstituted amino acids for purposes of stabilizing the alphahelix are described herein and include, for example, Aib. In someaspects, one, two, three, or more of the amino acids at positions 16,20, 21, and 24 of SEQ ID NO: 1 are substituted with an α,α-disubstitutedamino acid, e.g., Aib. In particular embodiments, the amino acid atposition 16 is Aib.

The analog which exhibits agonist activity at the GIP receptor cancomprise additional modifications, such as any of those describedherein. For instance, the amino acid modifications may increase ordecrease activity at one or both of the GLP-1 receptor and glucagonreceptor. The amino acid modifications may increase stability of thepeptide, e.g., increase resistance to DPP-IV protease degradation,stabilize the bond between amino acids 15 and 16. The amino acidmodifications may increase the solubility of the peptide and/or alterthe time of action of the analog at any of the GIP, glucagon, and GLP-1receptors. A combination of any of these types of modifications may bepresent in the analogs which exhibit agonist activity at the GIPreceptor.

Accordingly, in some aspects, the analog comprises the amino acidsequence of SEQ ID NO: 1 with one or more of: Gln at position 17, Ala atposition 18, Glu at position 21, Ile at position 23, and Ala or Cys atposition 24, or conservative amino acid substitutions thereof. In someaspects, the analog comprises a C-terminal amide in place of theC-terminal alpha carboxylate. In certain embodiments, the analogcomprises an amino acid substitution at position 1, position 2, orpositions 1 and 2, which substitution(s) achieve DPP-IV proteaseresistance. Suitable amino acid substitutions are described herein. Forexample, DMIA at position 1 and/or d-Ser or Aib at position 2. In someembodiments, the amino acid at position 2 is not D-serine.

Additionally or alternatively, the analog may comprise one or acombination of: (a) Ser at position 2 substituted with Ala; (b) Gln atposition 3 substituted with Glu or a glutamine analog; (c) Thr atposition 7 substituted with a Ile; (d) Tyr at position 10 substitutedwith Trp or an amino acid comprising an acyl or alkyl group which isnon-native to a naturally-occurring amino acid; (e) Lys at position 12substituted with Ile; (f) Asp at position 15 substituted with Glu; (g)Ser at position 16 substituted with Glu; (h) Gln at position 20substituted with Ser, Thr, Ala, Aib; (i) Gln at position 24 substitutedwith Ser, Thr, Ala, Aib; (j) Met at position 27 substituted with Leu orNle; (k) Asn at position 29 substituted with a charged amino acid,optionally, Asp or Glu; and (1) Thr at position 29 substituted with Glyor a charged amino acid, optionally, Asp or Glu.

With regard to the analogs which exhibit agonist activity at the GIPreceptor, the analog comprises an extension of 1-21 amino acids (e.g.,5-19, 7-15, 9-12 amino acids). The extension of the analog may compriseany amino acid sequence, provided that the extension is 1 to 21 aminoacids. In some aspects, the extension is 7 to 15 amino acids and inother aspects, the extension is 9 to 12 amino acids. In someembodiments, the extension comprises (i) the amino acid sequence of SEQID NO: 26 or 674, (ii) an amino acid sequence which has high sequenceidentity (e.g., at least 80%, 85%, 90%, 95%, 98%, 99%) with the aminoacid sequence of SEQ ID NO: 26 or 674, or (iii) the amino acid sequenceof (i) or (ii) with one or more conservative amino acid modifications.

In some embodiments, at least one of the amino acids of the extension isacylated or alkylated. The amino acid comprising the acyl or alkyl groupmay be located at any position of extension of the analog. In certainembodiments, the acylated or alkylated amino acid of the extension islocated at one of positions 37, 38, 39, 40, 41, or 42 (according to thenumbering of SEQ ID NO: 1) of the analog. In certain embodiments, theacylated or alkylated amino acid is located at position 40 of theanalog.

In exemplary embodiments, the acyl or alkyl group is an acyl or alkylgroup which is non-native to a naturally-occurring amino acid. Forexample, the acyl or alkyl group may be a C4 to C30 (e.g., C12 to C18)fatty acyl group or C4 to C30 (e.g., C12 to C18) alkyl. The acyl oralkyl group may be any of those discussed herein.

In some embodiments, the acyl or alkyl group is attached directly to theamino acid, e.g., via the side chain of the amino acid. In otherembodiments, the acyl or alkyl group is attached to the amino acid via aspacer (e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional spacer, a hydrophobic bifunctional spacer). In certainaspects, the spacer is 3 to 10 atoms in length. In some embodiments, theamino acid spacer is not γ-Glu. In some embodiments, the dipeptidespacer is not γ-Glu-γ-Glu.

Also, in exemplary embodiments, the amino acid to which the acyl oralkyl group is attached may be any of those described herein, including,for example, an amino acid of Formula I, II, or III. The amino acidwhich is acylated or alkylated may be a Lys, for example. Suitable aminoacids comprising an acyl or alkyl group, as well as suitable acyl groupsand alkyl groups, are described herein. See, for example, the teachingsunder the sections entitled Acylation and Alkylation.

In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 aminoacids) of the extension are positive-charged amino acids, e.g., aminoacids of Formula IV, such as, for example, Lys. As used herein, the term“positive-charged amino acid” refers to any amino acid,naturally-occurring or non-naturally occurring, comprising a positivecharge on an atom of its side chain at a physiological pH. In certainaspects, the positive-charged amino acids are located at any ofpositions 37, 38, 39, 40, 41, 42, and 43. In specific embodiments, apositive-charged amino acid is located at position 40.

In other instances, the extension is acylated or alkylated as describedherein and comprises 1-6 positive charged amino acids as describedherein.

In yet other embodiments, the analogs which exhibit agonist activity atthe GIP receptor comprises (i) SEQ ID NO: 1 with at least one amino acidmodification, (ii) an extension of 1 to 21 amino acids (e.g., 5 to 18, 7to 15, 9 to 12 amino acids)C-terminal to the amino acid at position 29of the analog, and (iii) an amino acid comprising an acyl or alkyl groupwhich is non-native to a naturally-occurring amino acid which is locatedoutside of the C-terminal extension (e.g., at any of positions 1-29). Insome embodiments, the analog comprises an acylated or alkylated aminoacid at position 10. In particular aspects, the acyl or alkyl group is aC4 to C30 fatty acyl or C4 to C30 alkyl group. In some embodiments, theacyl or alkyl group is attached via a spacer, e.g., an amino acid,dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobicbifunctional spacer). In certain aspects, the analog comprises an aminoacid modification which stabilizes the alpha helix, such as a saltbridge between a Glu at position 16 and a Lys at position 20, or analpha, alpha-disubstituted amino acid at any one, two, three, or more ofpositions 16, 20, 21, and 24. In specific aspects, the analogadditionally comprises amino acid modifications which confer DPP-IVprotease resistance, e.g., DMIA at position 1, Aib at position 2.Analogs comprising further amino acid modifications are contemplatedherein.

In certain embodiments, the analogs having GIP receptor activity exhibitat least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%)activity of native GIP at the GIP receptor. In some embodiments, theanalogs exhibit more than 20% (e.g., more than 50%, more than 75%, morethan 100%, more than 200%, more than 300%, more than 500%) activity ofnative GIP at the GIP receptor. In some embodiments, the analog exhibitsappreciable agonist activity at one or both of the GLP-1 and glucagonreceptors. In some aspects, the selectivity for these receptors (GIPreceptor and GLP-1 receptor and/or glucagon receptor) are within1000-fold. For example, the selectivity for the GLP-1 receptor of theanalogs having GIP receptor activity can be less than 500-fold,100-fold, within 50-fold, within 25 fold, within 15 fold, within 10fold) the selectivity for the GIP receptor and/or the glucagon receptor.

In accordance with some embodiments, the Class 3 glucagon relatedpeptide comprises the amino acid sequence of native glucagon (SEQ IDNO: 1) comprising the following modifications: Aib at position 2, Glu atposition 3, Lys at position 10, Glu at position 16, Gln at position 17,Ala at position 18, Lys at position 20, Glu at position 21, Ile atposition 23, Ala at position 24; wherein Lys at position 10 is acylatedwith a C14 or C16 fatty acid, and wherein the C-terminal carboxylate isreplaced with an amide. In a specific embodiment, this Class 3 glucagonrelated peptide is attached via a linker (L) to a GPCR ligand (Y).

In accordance with some embodiments, the Class 3 glucagon relatedpeptide comprises, consists essentially of, or consists of an amino acidsequence of any of SEQ ID NOs: 70-514, 517-534, or 554, optionally withup to 1, 2, 3, 4, or 5 further modifications that retain GLP-1 agonistand/or glucagon agonist activity. In certain embodiments, the Class 3glucagon related peptide comprises the amino acids of any of SEQ ID NOs:562-760. In some embodiments, the Class 3 glucagon related peptidecomprises the amino acid sequences of any of SEQ ID NOs: 1301-1421.

Class 4 Glucagon Related Peptides

In certain embodiments, Q is a Class 4 glucagon related peptide (see,e.g., International (PCT) Patent Application Publication No. WO2009/058662, incorporated herein by reference in its entirety.

All biological sequences referenced in the following section (SEQ IDNOs: 1301-1371) correspond to SEQ ID NOs: 1-71 in WO 2009/058662.

Activity

In accordance with some embodiments, Class 4 glucagon related peptidesare provided (hereafter referred to as “Class 4 peptides”). In certainaspects a Class 4 peptide is provided which has glucagon antagonistactivity. A glucagon antagonists would be used in any setting where thesuppression of glucagon agonism is desired. The most immediate andobvious use would be in the treatment of diabetes where glucagonantagonism has been demonstrated in pre-clinical models of hyperglycemiato yield a lowering of blood glucose. Glucagon antagonists can befurther modified to improve the biophysical stability and/or aqueoussolubility of the compounds while maintaining the antagonist activity ofthe parent compound. In certain aspects a Class 4 peptide is defined asa pure glucagon antagonist.

The term “glucagon antagonist” refers to a compound that counteractsglucagon activity or prevents glucagon function. For example, a glucagonantagonist exhibits at least 60% inhibition (e.g., at least 70%inhibition) and preferably, at least 80% inhibition, of the maximumresponse achieved by glucagon at the glucagon receptor. In someembodiments, the glucagon antagonist exhibits at least 90% inhibition ofthe maximum response achieved by glucagon at the glucagon receptor. In aspecific embodiment, the glucagon antagonist exhibits 100% inhibition ofthe maximum response achieved by glucagon at the glucagon receptor.Additionally, a glucagon antagonist at a concentration of about 1 μMexhibits less than about 20% of the maximum agonist activity achieved byglucagon at the glucagon receptor. In some embodiments, the glucagonantagonist exhibits less than about 10% of the maximum agonist activityachieved by glucagon at the glucagon receptor. In a specific embodiment,the glucagon antagonist exhibits less than about 5% of the maximumagonist activity achieved by glucagon at the glucagon receptor. In yetanother specific embodiment, the glucagon antagonist exhibits 0% of themaximum agonist activity achieved by glucagon at the glucagon receptor.

A “pure glucagon antagonist” is a glucagon antagonist that does notproduce any detected stimulation of glucagon or GLP-1 receptor activity,as measured by cAMP production using a validated in vitro model assay(see, e.g., WO 2009/058662). For example, a pure glucagon antagonistexhibits less than about 5% (e.g., less than about 4%, less than about3%, less than about 2%, less than about 1%, about 0%) of the maximumagonist activity achieved by glucagon at the glucagon receptor andexhibits less than about 5% (e.g., less than about 4%, less than about3%, less than about 2%, less than about 1%, and about 0%) of the maximumagonist activity achieved by GLP-1 at the GLP-1 receptor.

Accordingly, in some aspects, there is provided Class 4 peptides thatexhibit pure glucagon antagonist activity. In accordance with someembodiments the glucagon antagonist exhibits activity that reducesglucagon receptor glucagon-induced cAMP production by a maximum of atleast 50% when the glucagon receptor is contacted simultaneously with0.8 nM of glucagon and the glucagon antagonist, as measured by cAMPproduction in an in vitro assay. In some embodiments, the glucagonantagonist reduces glucagon receptor glucagon-induced cAMP production bya maximum amount of at least 80%.

Class 4 peptides are believed to be suitable for any use that haspreviously been described for glucagon antagonists. Accordingly, theClass 4 peptides described herein can be used to treat hyperglycemia, ortreat other metabolic diseases that result from high blood levels ofglucagon or high blood glucose levels. In accordance with someembodiments the patient to be treated using the Class 4 peptidesdisclosed herein is a domesticated animal, and in another embodiment thepatient to be treated is a human. Studies suggest that lack of glucagonsuppression in diabetic patients contributes to postprandialhyperglycemia in part via accelerated glycogenolysis. Analysis of bloodglucose during an Oral Glucose Tolerance Test (OGTT), and in thepresence or absence of somatostatin-induced glucagon suppression, hasshown a significant increase in glucose in subjects with higher glucagonlevels. Accordingly, the Class 4 peptides of the present invention canbe used to treat hyperglycemia, and are expected to be useful fortreating a variety of types of diabetes including diabetes mellitus typeI, diabetes mellitus type II, or gestational diabetes, eitherinsulin-dependent or non-insulin-dependent, and reducing complicationsof diabetes including nephropathy, retinopathy and vascular disease.

In some embodiments the terminal ten amino acids of Exendin-4 (i.e. thesequence of SEQ ID NO: 1319 (GPSSGAPPPS)) are linked to the carboxyterminus of a Class 4 peptide. These fusion proteins are anticipated tohave pharmacological activity for suppressing appetite and inducingweight loss/weight maintenance. In accordance with some embodiments theClass 4 peptides disclosed herein can be further modified to include theamino acid sequence of SEQ ID NO: 1319 (GPSSGAPPPS) linked to amino acid24 of the Class 4 peptide of SEQ ID NO: 1342 and administered toindividuals to induce weight loss or assist in weight maintenance. Moreparticularly, the Class 4 peptide comprises a sequence selected from thegroup consisting of SEQ ID NO: 1302, SEQ ID NO: 1303, SEQ ID NO: 1304SEQ ID NO: 1305, SEQ ID NO: 1306, SEQ ID NO: 1307, SEQ ID NO: 1308, SEQID NO: 1336, SEQ ID NO: 1339, SEQ ID NO: 1340 SEQ ID NO: 1341, SEQ IDNO: 1342, SEQ ID NO: 1343 and SEQ ID NO: 1344 and further comprising theamino acid sequence of SEQ ID NO: 1319 (GPSSGAPPPS) linked to amino acid24 of the Class 4 peptide is used to suppress appetite and inducingweight loss/weight maintenance. In some embodiments the administeredClass 4 peptide comprises the sequence of SEQ ID NO: 1346 or SEQ ID NO:1347.

Such methods for reducing appetite or promoting loss of body weight areexpected to be useful in reducing body weight, preventing weight gain,or treating obesity of various causes, including drug-induced obesity,and reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases.

The Class 4 peptides of the invention may be administered alone or incombination with other anti-diabetic or anti-obesity agents.Anti-diabetic agents known in the art or under investigation includeinsulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide(Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide(Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride(Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide(Prandin) or nateglinide (Starlix); biguanides such as metformin(Glucophage) or phenformin; thiazolidinediones such as rosiglitazone(Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or otherPPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydratedigestion, such as miglitol (Glyset), acarbose (Precose/Glucobay);exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4)inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependentglucose transporter 1) inhibitors; or FBPase (fructose1,6-bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to xenical (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

The Class 4 peptides of the present invention can also be administeredto patients suffering from catabolic wasting. It is estimated that overhalf of cancer patients experience catabolic wasting which ischaracterized by unintended and progressive weight loss, weakness, andlow body fat and muscle. The syndrome is equally common in AIDS patientsand can also be present in bacterial and parasitic diseases, rheumatoidarthritis, and chronic diseases of the bowel, liver, lungs, and heart.It is usually associated with anorexia and can manifest as a conditionin aging or as a result of physical trauma. Catabolic wasting is asymptom that diminishes the quality of life, worsens the underlyingcondition, and is a major cause of death. Applicants anticipate that theClass 4 peptides disclosed herein can be administered to patients totreat catabolic wasting.

Pharmaceutical compositions comprising the Class 4 peptides disclosedherein can be formulated and administered to patients to using standardpharmaeuctically acceptable carriers and routes of administration knownto those skilled in the art. Accordingly the present disclosure alsoencompasses pharmaceutical compositions comprising one or more of theClass 4 peptides disclosed herein in combination with a pharmaceuticallyacceptable carrier. The pharmaceutical compositions may comprise theClass 4 peptides as the sole pharmaceutically active component, or theClass 4 peptides can be combined with one or more additional activeagents. In accordance with some embodiments a composition is providedcomprising a Class 4 peptide of the present invention and a compoundthat activates the GLP-1 receptor (such as GLP-1, a GLP-1 analog, anexendin-4 analog, or derivatives thereof). In accordance with someembodiments a composition is provided comprising a Class 4 peptide ofthe present invention and insulin or an insulin analog. Alternatively, acomposition provided for inducing weight loss or preventing weight gaincan be provided that comprises the sequence of SEQ ID NO: 1342 furthercomprising the amino acid sequence of SEQ ID NO: 1319 (GPSSGAPPPS)linked to amino acid 24 of SEQ ID NO: 1342, and an anti-obesity peptide.Suitable anti-obesity peptides include those disclosed in U.S. Pat. Nos.5,691,309, 6,436,435 or US Patent application 20050176643, andincluding, but not limited to GLP-1, GIP (Gastric InhibitoryPolypeptide), MP1, PYY, MC-4, Leptin.

Class 4 Peptide Structure

In some embodiments Class 4 glucagon related peptides are providedwherein the normally occurring aspartic acid at position nine (ofglucagon, SEQ ID NO: 1301) has been substituted with glutamic acid or acysteic acid-based derivative. More particularly, deletion of the firstamino acid (des-His) and substitution of the aspartic acid at position 9with glutamic acid, in some aspects, produces a Class 4 peptide. Class 4glucagon related peptides having sulfonic acid substituents substitutedat amino acid position nine of glucagon perform similarly to thecarboxylic acid-based amino acids but with a few critical differences inrelation to physical properties such as solubility. Homocysteic acid(hCysSO3) when substituted for the isosteric glutamic acid at positionnine in the conventional des-His, Glu9 Class 4 peptide retains a partialantagonist and weak agonist.

In some embodiments there is provided a Class 4 peptide wherein thefirst two to five amino acids are removed, and position 9 (according tothe numbering of SEQ ID NO: 1301) is replaced with hCys(SO3),homoglutamic acid, (3-homoglutamic acid, or an alkylcarboxylatederivative of cysteine having the structure of:

wherein X5 is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl, provides acompound that performs as a hormonal antagonist that is highly specific,potent and without contaminating agonist properties.

In accordance with some embodiments a Class 4 peptide is provided thatcomprises a glucagon peptide modified, relative to the wild typesequence of SEQ ID NO: 1301, by the deletion of two to five amino acidresidues from the N-terminus and substitution of the aspartic acidresidue at position nine of the native protein with a glutamic acid,homoglutamic acid, β-homoglutamic acid, a sulfonic acid derivative ofcysteine, or an alkylcarboxylate derivative of cysteine having thestructure of:

wherein X₅ is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl.

In one specific embodiment, the Class 4 peptide comprising the deletionof two to five amino acid residues from the N-terminus and substitutionof the Asp at position 9 of the native glucagon, is further modified byup to three amino acid modifications. For example, the Class 4 peptidemay comprise one, two, or three conservative amino acid modifications.Alternatively or additionally, the Class 4 peptide may comprise one ormore amino acid modifications selected from the group consisting of:

A. substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1301), or the N- orC-terminal amino acid of the Class 4 peptide with an amino acidcovalently attached to an acyl group or alkyl group via an ester, ether,thioether, amide, or alkyl amine linkage;

B. substitution of one or two amino acids at positions 16, 17, 20, 21,and 24 (according to the amino acid numbering of SEQ ID NO: 1301), orthe N- or C-terminal amino acid of the Class 4 peptide with an aminoacid selected from the group consisting of: Cys, Lys, ornithine,homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acidof the group is covalently bonded to a hydrophilic moiety;

C. addition of an amino acid covalently bonded to a hydrophilic moietyto the N- or C-terminus of the Class 4 peptide;

D. substitution of Asp at position 15 (according to the numbering of SEQID NO: 1301) with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid;

E. substitution of Ser at position 16 (according to the numbering of SEQID NO: 1301) with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid;

F. substitution with Aib at one or more of positions 16, 20, 21, and 24according to the amino acid numbering of SEQ ID NO: 1301;

G. deletion of the amino acid at position 29 or the amino acids atpositions 28 and 29, according to the numbering of SEQ ID NO: 1301;

H. substitution of each or both of the Asn at position 28 and the Thr atposition 29 (according to the amino acid numbering of SEQ ID NO: 1301)with charged amino acids; and/or addition of one to two charged aminoacids at the C-terminus of SEQ ID NO: 1301;

I. substitution of the Met at position 27 (according to the numbering ofSEQ ID NO: 1301) with Leu or norleucine;

J. addition of a peptide having the amino acid sequence of any of SEQ IDNOs: 19-21 and 53 to the C-terminus of SEQ ID NO: 1301; wherein Thr atposition 29 (according to the numbering of SEQ ID NO: 1301) is Thr orGly; and

K. replacement of the C-terminal carboxylate with an amide or ester.

In a specific embodiment, the Class 4 peptide comprises an amino acidmodification of A, B, or C, as described above, or a combinationthereof. In yet another specific embodiment, the Class 4 peptide furthercomprises an amino acid modification of any of D to K as describedabove, or a combination thereof, in addition to the amino acidmodification(s) of A, B, and/or C.

In some embodiments the Class 4 peptide comprises a glucagon peptide,wherein the first 5 amino acids have been removed from the N-terminus,and the remaining N-terminal amino group has been replaced with ahydroxyl group (the “PLA6 analog”), producing the peptide of SEQ ID NO:1339. Applicants have found that substitution of phenyl-lactic acid forphenylalanine in Class 4 peptide analogs that have the first five aminoacids deleted and substitution of a glutamic acid at position 9(relative to native glucagon) further enhances the potency of thoseClass 4 peptide analogs.

In some embodiments the Class 4 peptide peptide of SEQ ID NO: 1339 isfurther modified by substituting the aspartic acid residue at positionfour (position 9 of the native glucagon) with an amino acid of thegeneral structure:

wherein X₆ is C1-C3 alkyl, C2-C3 alkene or C2-C3 alkynyl, and in someembodiments X6 is C1-C3 alkyl, and in another embodiment X6 is C2 alkyl.In some embodiments the Class 4 peptide comprises a glucagon peptide,wherein the first 5 amino acids have been removed from the N-terminus,and the aspartic acid residue at position four (position 9 of the nativeglucagon) has been substituted with cysteic acid or homocysteic acid. Insome embodiments the Class 4 peptide comprises a glucagon peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1339, SEQ ID NO: 1307 and SEQ ID NO: 1308. In someembodiments the Class 4 peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1308, wherein the aminoacid at position four is homocysteic acid.

In another embodiment, the Class 4 peptide of SEQ ID NO: 1339 is furthermodified by substituting the aspartic acid residue at position four(position 9 of the native glucagon) with glutamic acid, homoglutamicacid, β-homoglutamic acid, or an alkylcarboxylate derivative of cysteinehaving the structure of:

wherein X₅ is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl. In aspecific embodiment, X₅ is C1 or C2 alkyl.

However, applicants have discovered that with the substitution of theN-terminal phenylalanine with PLA in a des1-5 glucagon analog (i.e., aglucagon analog having the first five amino acids deleted), furthersubstitution of the native aspartic acid residue at position four(position 9 of the native glucagon) is not required to produce an analogthat exhibits pure antagonism. This result is surprising in light of theprior art teachings that the native aspartic acid residue at positionfour must substituted to produce high affinity and potent antagonists ofglucagon (2-29) analogs. The use of the PLA substitution improves therelative potency of the Asp9 analog to a point comparable to that of theGlu9 and hCys(SO₃H)9 analogs.

Substitution of the phenylalanine residue with other phenylalanineanalogs, including 3,4-2F-phenylalnine (3,4-2F-Phe), 2-naphthyalanine(2-Nal), N-acyl-phenylalanine (Ac-Phe), alpha-methylhydrocinnamic acid(MCA) and benzylmalonic acid (BMA) did not perform as potently as thePLA substitution.

Substituting PLA at sites other than at position six (according to theamino acid numbering of native glucagon), including at positions 4 and 5reveals that the PLA6 analog is an appreciably more potent antagonistthan glucagon analogs having a slightly extended N-terminus. The presentinvention also includes analogs wherein the N-terminal amino group issubstituted with an acylated and alkylated “O-terminal” peptides.

Furthermore, the PLA6 substitution not only increases the potency of theantagonist but also serves a critical role in pegylation. The PLA6analogs can be selectively pegylated without restoration of glucagonagonism. In the absence of the PLA substitution, pegylation of theanalog surprisingly induces glucagon agonism. This glucagon agonism isnot seen in the pegylated PLA6 analogs. Several sites for pegylationwere investigated including positions 3, 6 and 19 (positions 8, 11 and19 of native glucagon) and at the N-terminal amino acid residue. In someembodiments the pegylation is at position 19 (position 24 of nativeglucagon) as that site exhibits the most potent and selective glucagonantagonism.

In some embodiments, the Class 4 peptide comprises the general structureof A-B-C, wherein A is selected from the group consisting of:

(i) phenyl lactic acid (PLA);

(ii) an oxy derivative of PLA;

(iii) a peptide of 2 to 6 amino acids in which two consecutive aminoacids of the peptide are linked via an ester or ether bond; B representsamino acids i to 26 of SEQ ID NO: 1301, wherein i is 3, 4, 5, 6, or 7,optionally comprising one or more amino acid modifications selected fromthe group consisting of:

(iv) Asp at position 9 (according to the amino acid numbering of SEQ IDNO: 1301) is substituted with a Glu, a sulfonic acid derivative of Cys,homoglutamic acid, β-homoglutamic acid, or an alkylcarboxylatederivative of cysteine having the structure of:

wherein X5 is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl.

(v) substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1301) with an aminoacid covalently attached to an acyl or alkyl group via an ester, ether,thioether, amide, or alkyl amine linkage;

(vi) substitution of one or two amino acids at positions 16, 17, 20, 21,and 24 (according to the amino acid numbering of SEQ ID NO: 1301) withan amino acid selected from the group consisting of: Cys, Lys,ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein theamino acid of the group is covalently attached to a hydrophilic moiety;

(vii) Asp at position 15 (according to the numbering of SEQ ID NO: 1301)is substituted with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid;

(viii) Ser at position 16 (according to the numbering of SEQ ID NO:1301) is substituted with cysteic acid, glutamic acid, homoglutamicacid, and homocysteic acid;

(ix) substitution with Aib at one or more of positions 16, 20, 21, and24 according to the amino acid numbering of SEQ ID NO: 1301;

and C is selected from the group consisting of:

(x) X;

(xi) X-Y;

(xii) X-Y-Z; and

(xiii) X-Y-Z-R10,

wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z isThr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine(Ac-Phe), or a charged amino acid; wherein R10 is selected from a groupconsisting of SEQ ID NOs: 1319-1321 and 1353; and

(xiv) any of (x) to (xiii) in which the C-terminal carboxylate isreplaced with an amide.

In a specific aspect, the Class 4 peptide comprises an oxy derivative ofPLA. As used herein “oxy derivative of PLA” refers to a compoundcomprising a modified structure of PLA in which the hydroxyl group hasbeen replaced with O—R₁₁, wherein R₁₁ is a chemical moiety. In thisregard, the oxy derivative of PLA can be, for example, an ester of PLAor an ether of PLA.

Methods of making oxy derivatives of PLA are known in the art. Forexample, when the oxy derivative is an ester of PLA, the ester may beformed by upon reaction of the hydroxyl of PLA with a carbonyl bearing anucleophile. The nucleophile can be any suitable nucleophile, including,but not limited to an amine or hydroxyl. Accordingly, the ester of PLAcan comprise the structure of Formula IV:

wherein R₇ is an ester formed upon reaction of the hydroxyl of PLA witha carbonyl bearing a nucleophile.

The carbonyl bearing a nucleophile (which reacts with the hydroxyl ofPLA to form an ester) can be, for example, a carboxylic acid, acarboxylic acid derivative, or an activated ester of a carboxylic acid.The carboxylic acid derivative can be, but is not limited to, an acylchloride, an acid anhydride, an amide, an ester, or a nitrile. Theactivated ester of a carboxylic acid can be, for example,N-hydroxysuccinimide (NHS), tosylate (Tos), a carbodiimide, or ahexafluorophosphate. In some embodiments, the carbodiimide is1,3-dicyclohexylcarbodiimide (DCC), 1,1′-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or1,3-diisopropylcarbodiimide (DICD). In some embodiments, thehexafluorophosphate is selected from a group consisting ofhexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), ando-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU).

Methods of making ethers from reaction with a hydroxyl group (e.g., thehydroxyl of PLA) also are known in the art. For example, the hydroxylgroup of PLA may be reacted with a halogenated alkyl or tosylated alkylalcohol to form an ether bond.

Generally, the chemical moiety of R₁₁ is one which does not decrease theactivity of the Class 4 peptide. In some embodiments, the chemicalmoiety enhances the activity, stability, and/or solubility of the Class4 peptide.

In a specific embodiment, the chemical moiety bound to PLA via anoxygen-containing bond (e.g., via an ester or ether bond) is a polymer(e.g., a polyalkylene glycol), a carbohydrate, an amino acid, a peptide,or a lipid, e.g., a fatty acid or a steroid.

In a specific embodiment, the chemical moiety is an amino acid, which,optionally, is a part of a peptide, such that Formula IV is adepsipeptide. In this regard, PLA may be at a position other than theN-terminal amino acid residue of the Class 4 peptide, such that theClass 4 peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more)amino acids N-terminal to the PLA residue. For example, the Class 4peptide can comprise PLA at position n, wherein n is 2, 3, 4, 5, or 6 ofthe Class 4 peptide.

The amino acids N-terminal to the PLA residue may be synthetic ornaturally-occurring. In a specific embodiment, the amino acids which areN-terminal PLA are naturally-occurring amino acids. In some embodiments,the amino acids which are N-terminal to PLA are the N-terminal aminoacids of native glucagon. For example, the Class 4 peptide can compriseat the N-terminus the amino acid sequence of any of SEQ ID NOs:1354-1358, wherein PLA is linked to threonine via an ester bond:

His-Ser-Gln-Gly-Thr-PLA SEQ ID NO: 1354 Ser-Gln-Gly-Thr-PLASEQ ID NO: 1355 Gln-Gly-Thr-PLA SEQ ID NO: 1356 Gly-Thr-PLASEQ ID NO: 1357 Thr-PLA SEQ ID NO: 1358

In an alternative embodiment, one or more of the N-terminal amino acidsmay be substituted with an amino acid other than the amino acid ofnative glucagon. For example, when the Class 4 peptide comprises PLA asthe amino acid at position 5 or 6, the amino acid at position 1 and/orposition 2 may be an amino acid which reduces susceptibility to cleavageby dipeptidyl peptidase IV. More particularly, in some embodiments,position 1 of the Class 4 peptide is an amino acid selected from thegroup consisting of D-histidine, alpha, alpha-dimethyl imidiazole aceticacid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazoleacetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. More particularly, in some embodiments, position 2 ofthe antagonist peptide is an amino acid selected from the groupconsisting of D-serine, D-alanine, valine, glycine, N-methyl serine,N-methyl alanine, and aminoisobutyric acid (Aib). Also, for example,when the Class 4 peptide comprises PLA as the amino acid at position 4,5, or 6, the amino acid at position 3 of the Class 4 peptide may beglutamic acid, as opposed to the native glutamine residue of nativeglucagon. In an exemplary embodiment of the invention, the Class 4peptide comprises at the N-terminus the amino acid sequence of any ofSEQ ID NOs: 1359-1361.

With respect to the Class 4 peptides comprising a compound of FormulaIV, the polymer may be any polymer, provided that it can react with thehydroxyl group of PLA. The polymer may be one that naturally or normallycomprises a carbonyl bearing a nucleophile. Alternatively, the polymermay be one which was derivatized to comprise the carbonyl bearing thecarbonyl. The polymer may be a derivatized polymer of any of:polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

The polymer can be a biodegradable polymer, including a syntheticbiodegradable polymer (e.g., polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone)), and a naturalbiodegradable polymer (e.g., alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins (e.g., zein and other prolamines and hydrophobic proteins)), aswell as any copolymer or mixture thereof. In general, these materialsdegrade either by enzymatic hydrolysis or exposure to water in vivo, bysurface or bulk erosion.

The polymer can be a bioadhesive polymer, such as a bioerodible hydrogeldescribed by H. S. Sawhney, C. P. Pathak and J. A. Hubbell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer. Suitablewater-soluble polymers are known in the art and include, for example,polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel),hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose,hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose,hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose(Ethocel), hydroxyethyl cellulose, various alkyl celluloses andhydroxyalkyl celluloses, various cellulose ethers, cellulose acetate,carboxymethyl cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, vinyl acetate/crotonic acid copolymers,poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylicacid copolymers, polymethacrylic acid, polymethylmethacrylate, maleicanhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium andcalcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers,carboxypolymethylene, carboxyvinyl polymers, polyoxyethylenepolyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,carboxymethylamide, potassium methacrylate divinylbenzene co-polymer,polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, andcombinations thereof.

In a specific embodiment, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

The carbohydrate may be any carbohydrate provided that it comprises oris made to comprise a carbonyl with an alpha leaving group. Thecarbohydrate, for example, may be one which has been derivatized tocomprise a carbonyl with an alpha leaving group. In this regard, thecarbohydrate may be a derivatized form of a monosaccharide (e.g.,glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose,maltose), an oligosaccharide (e.g., raffinose, stachyose), apolysaccharide (a starch, amylase, amylopectin, cellulose, chitin,callose, laminarin, xylan, mannan, fucoidan, galactomannan.

With respect to the Class 4 peptides comprising a compound of FormulaIV, the lipid may be any lipid comprising a carbonyl with an alphaleaving group. The lipid, for example, may be one which is derivatizedto comprise the carbonyl. In this regard, the lipid, may be a derivativeof a fatty acid (e.g., a C4-C30 fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide. In some embodiments, the lipid is a oil, wax, cholesterol,sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, aphospholipid.

In some embodiments, R₇ has a molecular weight of about 100 kDa or less,e.g., about 90 kDa or less, about 80 kDa or less, about 70 kDa or less,about 60 kDa or less, about 50 kDa or less, about 40 kDa or less.Accordingly, R7 can have a molecular weight of about 35 kDa or less,about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about15 kDa or less, about 10 kDa or less, about 5 kDa or less, or about 1kDa.

In an alternative embodiment, the Class 4 peptide comprises as A, apeptide of 2 to 6 amino acids in which two consecutive amino acids ofthe peptide are linked via an ester or ether bond. The ester or etherbond may be, e.g., between amino acids 2 and 3, 3 and 4, 4 and 5, or 5and 6. Optionally the peptide may be further modified by covalentlinkage to another chemical moiety including linkage to a polymer (e.g.a hydrophilic polymer), alkylation, or acylation.

With regard to the Class 4 peptide comprising the general structureA-B-C, B represents amino acids of native glucagon, e.g., i to 26 of SEQID NO: 1301, wherein i is 3, 4, 5, 6, or 7, optionally comprising one ormore amino acid modifications. In a specific embodiment, B representsamino acids 7 to 26 of SEQ ID NO: 1301, optionally further modified.

In some embodiments, B is modified by up to three amino acidmodifications. For example, B, which represents native amino acidsequence of SEQ ID NO: 1301 is modified by one or more conservativeamino acid modifications.

In another embodiment, B comprises one or more amino acid modificationsselected from the group consisting of (iv) to (ix), as described herein.In a specific embodiment, B comprises one or both of the amino acidmodifications (v) and (vi). In a further specific embodiment, Bcomprises one or a combination of amino acid modifications selected fromthe group consisting of (iv), (vii), (viii), and (ix), in addition to(v) and (vi).

In another specific embodiment, the Class 4 peptide comprises one ormore charged amino acids at the C-terminus. For example, Y and/or Z canbe a charged amino acid, e.g., Lys, Arg, His, Asp, and Glu. In yetanother embodiment, the Class 4 peptide comprises one to two chargedamino acids (e.g., Lys, Arg, His, Asp, and Glu)C-terminal to Z. In aspecific aspect, Z followed by one to two charged amino acids does notcomprise R10.

The Class 4 peptide in some embodiments comprises a hydrophilic moietycovalently bound to an amino acid residue of the Class 4 peptide, asdescribed herein. For example, the Class 4 peptide can comprise ahydrophilic moiety covalently attached to an amino acid at position 1,16, 20, 21, or 24 according to the numbering of SEQ ID NO: 1301. Inanother embodiment, the hydrophilic moiety is attached to the C-terminalamino acid of the Class 4 peptide, which in some cases, is 1 or 11 aminoacids C-terminal to Z. In yet another embodiment, the hydrophilic moietyis attached to PLA, when A is PLA, PLA-Phe, or PLA-Thr-Phe, wherein PLAis modified to comprise the hydrophilic moiety. In another embodiment,an amino acid comprising a hydrophilic moiety is added to the N- orC-terminus of the Class 4 peptide. In another embodiment, the Class 4peptide comprises an acyl group or alkyl group as described herein. Forexample, the acylation or alkylation can occur off the side chain of theamino acid at position 10, 20, or 24, according to the numbering of SEQID NO: 1301. In an alternative embodiment, the acylation or alkylationoccurs off the side chain of the C-terminal amino acid of the Class 4peptide, which in some cases, is 1 or 11 amino acids C-terminal to Z. Inyet another embodiment, when A is PLA, PLA-Phe, or PLA-Thr-Phe, the PLAis modified to comprise an acyl or alkyl group.

Exemplary Embodiments

The Class 4 peptide may comprise any amino acids, synthetic or naturallyoccurring, provided that at least two consecutive amino acids of thepeptide are linked via an ester or ether bond. In a specific embodiment,the peptide comprises amino acids of native glucagon. For example, thepeptide can comprise j to 6 of native glucagon (SEQ ID NO: 1301),wherein j is 1, 2, 3, 4, or 5. Alternatively, the peptide can comprisean amino acid sequence based on the N-terminus of SEQ ID NO: 1301 withone or more amino acid modifications. The amino acid at position 1and/or position 2 may be an amino acid which reduces susceptibility tocleavage by dipeptidyl peptidase IV. For instance, the peptide cancomprise at position 1 of the Class 4 peptide an amino acid selectedfrom the group consisting of D-histidine, alpha, alpha-dimethylimidiazole acetic acid (DMIA), N-methyl histidine, alpha-methylhistidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine,acetyl-histidine and homo-histidine. More particularly, in someembodiments, position 2 of the antagonist peptide is an amino acidselected from the group consisting of D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid(Aib). Also, for example, the amino acid at position 3 of the Class 4peptide may be glutamic acid, as opposed to the native glutamine residueof native glucagon. Accordingly, the Class 4 peptide can comprise anamino acid sequence of:

Xaa1-Xaa2-Xaa3-Thr-Gly-Phe; (SEQ ID NO: 1368) Xaa2-Xaa3-Thr-Gly-Phe;(SEQ ID NO: 1369) or Xaa3-Thr-Gly-Phe; (SEQ ID NO: 1370)wherein Xaa1 is selected from a group consisting of: His, D-histidine,alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine; Xaa2 isselected from a group consisting of: Ser, D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid(Aib); and Xaa3 is Gln or Glu.

The present invention also encompasses embodiments wherein theC-terminal amino acid of the Class 4 peptides have an amide groupsubstituting for the carboxylic acid group that is present on the nativeamino acid.

In some embodiments, wherein the Class 4 peptide is PEGylated, the Class4 peptide comprises the shortened glucagon peptides, specifically 6-29where the “N-terminal” amino acid is PLA (phenyl-lactic acid). Suchglucagon derivatives exhibit unique virtues. They are more potentpeptides than those with the native N-terminal phenylalanine and theysuppress any glucagon agonism that results from pegylation, somethingnot seen with the native phenylalanine. Finally, while the currentliterature establishes that a substitution of the native aspartic acidat position 9 is required for antagonist activity, applicants havediscovered the surprising result that such a substitution is no longerrequired in the PLA6-(6-29) glucagon analogs.

In some embodiments an amino acid of the Class 4 peptide is substitutedwith at least one cysteine residue, wherein the side chain of thecysteine residue is further modified with a thiol reactive reagent,including for example, maleimido, vinyl sulfone, 2-pyridylthio,haloalkyl, and haloacyl. These thiol reactive reagents may containcarboxy, keto, hydroxyl, and ether groups as well as other hydrophilicmoieties such as polyethylene glycol units. In an alternativeembodiment, an amino acid of the Class 4 peptide is substituted withlysine, and the side chain of the substituting lysine residue is furthermodified using amine reactive reagents such as active esters(succinimido, anhydride, etc) of carboxylic acids or aldehydes ofhydrophilic moieties such as polyethylene glycol. In accordance withsome embodiments the lysine residue corresponding to position 12 of thenative peptide is substituted with arginine and a single lysinesubstitution is inserted for one of the amino acids corresponding toposition 1, 16, 17, 20, 21, 24 or 29 of the native peptide, or a lysineis added to the N- or C-terminus of the Class 4 peptide.

In another embodiment the methionine residue corresponding to position27 of the native peptide is changed to leucine or norleucine to preventoxidative degradation of the peptide.

In some embodiments, the Class 4 peptides described herein are furthermodified by truncation or deletion of one or two amino acids of theC-terminus of the glucagon peptide (i.e., truncation of the amino acidat position 29 or at positions 28 and 29 of native glucagon) withoutaffecting activity and/or potency at the glucagon receptor. In thisregard, the Class 4 peptide described herein can, for example, consistessentially of or consist of amino acids 1-27, 1-28, 2-27, 2-28, 3-27,3-28, 4-27, 4-28, 5-27, 5-28, 6-27, or 6-28 of the native glucagonpeptide (SEQ ID NO: 1301) with one or more modifications resulting inClass 4 peptidic activity as described herein.

The presently disclosed Class 4 peptides also encompass amino acidsubstitutions at positions that are known not to be critical to thefunction of the glucagon peptide. In some embodiments the substitutionsare conservative amino acid substitutions at one, two or three positionsselected from the group consisting of 2, 5, 6, 7, 8, 9, 12, 13, 14, 15,16, 19, 22, 23 or 24 of SEQ ID NO: 1339. In some embodiments the Class 4peptide comprises a derivative peptide of SEQ ID NO: 1342 wherein theglucagon peptide comprises a further amino acid substitution relative toSEQ ID NO: 1342 at one to three amino acid positions selected frompositions 2, 5, 6, 8, 9, 12, 13 and 14. In some embodiments thesubstitutions at positions 2, 5, 6, 8, 9, 12, 13 and 14 of SEQ ID NO:1342 are conservative amino acid substitutions. In some embodiments theamino acids corresponding to positions 16, 17, 20, 21, 24 or 29 of thenative peptide, and more particularly at position 21 and/or 24 aresubstituted with cysteine or lysine, wherein a PEG chain is covalentlyattached to the substituted cysteine or lysine residue.

In accordance with some embodiments the modified Class 4 peptidecomprises two or more polyethylene glycol chains covalently bound to thepeptide wherein the total molecular weight of the glucagon chains isabout 1,000 to about 5,000 Daltons. In some embodiments the pegylatedClass 4 peptide comprises a peptide selected from the group consistingof SEQ ID NO: 1312, and SEQ ID NO: 1322, wherein said peptide comprise apolyethylene glycol chain linked to the amino acid at positions 11 and19 and the combined molecular weight of the two PEG chains is about1,000 to about 5,000 Daltons.

In accordance with some embodiments a Class 4 peptide is providedcomprising a modified glucagon peptide selected from the groupconsisting of:

(SEQ ID NO: 1309) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Asn- Thr-R₂,(SEQ ID NO: 1310) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Gln-Trp-Leu-Xaa-Asn- Thr-R₂,(SEQ ID NO: 1311) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Xaa-Trp-Leu-Xaa-Asn- Thr-R₂ and(SEQ ID NO: 1312) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Xaa-Trp-Leu-Xaa-Asn- Thr-R₂,wherein Xaa at position 4=aspartic acid, glutamic acid, cysteic acid orhomocysteic acid, Xaa at position 7=Lys or Arg, Xaa at position 10 isaspartic acid, cysteic acid, glutamic acid, homoglutamic acid andhomocysteic acid; Xaa at position 11 is Ser, Lys, Cys, Orn, homocysteineor acetyl phenylalanine, Xaa at position 16 is Asp, Lys, Cys, Orn,homocysteine or acetyl phenylalanine a and Xaa at position 19 is Gln,Lys, Cys, Orn, homocysteine and acetyl phenylalanine, Xaa at position22=Met, Leu or Nle, R1 is OH or NH₂, and R₂ is COOH or CONH₂, whereinthe peptide is pegylated at position 11 for SEQ ID NO: 1309, at position16 for SEQ ID NO: 1310, position 19 for SEQ ID NO: 1311 and at positions16 and 19 of SEQ ID NO: 1312, with the proviso that when Xaa at position4=aspartic acid then R₁ is OH. In accordance with some embodiments thepeptide comprises the sequence of SEQ ID NO: 1309, SEQ ID NO: 1310 orSEQ ID NO: 1311, wherein R₁ is OH and R₂ is CONH₂. In some embodimentsthe peptide comprises the sequence of SEQ ID NO: 1309, SEQ ID NO: 1310or SEQ ID NO: 1311, wherein R₁ is OH, R₂ is CONH₂ and the amino acid atposition 4 is aspartic acid, and in a further embodiment such peptidescomprise a carboxy terminal extension comprising the sequence of SEQ IDNO: 1319.

In accordance with some embodiments the peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 1309, SEQ ID NO: 1310,SEQ ID NO: 1313, SEQ ID NO: 1314, and SEQ ID NO: 1316, wherein thepeptide is pegylated at position 11 for SEQ ID NO: 1309 and SEQ ID NO:1313, pegylated at position 16 for SEQ ID NO: 1310, and pegylated atposition 19 for SEQ ID NO: 1310 and SEQ ID NO: 1314. In some embodimentsthe glucagon agonist comprises the peptide of SEQ ID NO: 1313 or SEQ IDNO: 1314. In some embodiments the C-terminal amino acid of the Class 4peptides disclosed herein have an amide group in place of the carboxylicacid group that is present on the native amino acid. In accordance withsome embodiments the Class 4 peptide comprises the sequence of SEQ IDNO: 1318.

In accordance with some embodiments, a Class 4 peptide is providedwherein a plasma protein has been covalently linked to an amino acidside chain of the peptide to improve the solubility, stability and/orpharmacokinetics of the glucagon peptide. For example, serum albumin canbe covalently bound to the Class 4 peptides presented herein. In someembodiments the plasma protein is covalently bound to an amino acidcorresponding to position 16, 17, 20, 21, 24 or 29 of the nativeglucagon peptide. More particularly, in some embodiments the plasmaprotein is bound to an amino acid corresponding to position 16 or 24 ofthe native glucagon peptide, wherein the Class 4 peptide comprises thesequence of SEQ ID NO: 1303, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ IDNO: 1306, SEQ ID NO: 1307, SEQ ID NO: 1308, SEQ ID NO: 1309, SEQ ID NO:1311, SEQ ID NO: 1312, SEQ ID NO: 1322, SEQ ID NO: 1323, SEQ ID NO:1324, SEQ ID NO: 1325, SEQ ID NO: 1326, SEQ ID NO: 1327, SEQ ID NO:1328, SEQ ID NO: 1336 and SEQ ID NO: 1339. In some embodiments the Class4 peptide comprises a peptide selected from the group consisting of SEQID NO: 1309, SEQ ID NO: 1310, SEQ ID NO: 1311 and SEQ ID NO: 1312.

In accordance with some embodiments, a Class 4 peptide is providedwherein a linear amino acid sequence representing the Fc portion of animmunoglobin molecule has been covalently linked to an amino acid sidechain of a Class 4 peptide disclosed herein to improve the solubility,stability and/or pharmacokinetics of the glucagon peptide. For example,the amino acid sequence representing the Fc portion of an immunoglobinmolecule can be covalently bound to position 11, 12, 15, 16, 19, 21 or24 of the glucagon peptide of SEQ ID NO: 1307, SEQ ID NO: 1339, or aglucagon analog thereof. In some embodiments the Fc peptide iscovalently bound to position 11 or 19 of the Class 4 peptide of SEQ IDNO: 1306, SEQ ID NO: 1307, SEQ ID NO: 1308 or SEQ ID NO: 1336. The Fcportion is usually isolated from IgG, but the Fc peptide fragment fromany immunoglobin should function equivalently. In some embodiments theglucagon peptide is selected from the group consisting of SEQ ID NO:1303, SEQ ID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1307 SEQ ID NO: 1308,and SEQ ID NO: 1339, wherein the Fc portion is linked to thecorresponding position of 16, 17, 20, 21, 24 or 29 of the nativeglucagon peptide. In some embodiments the Class 4 peptide comprises aglucagon peptide selected from the group consisting of SEQ ID NO: 1309,SEQ ID NO: 1310, SEQ ID NO: 1311 and SEQ ID NO: 1312, wherein the Fcpeptide is bound to the side chain of the amino acid located at position11, 16 or 19 of SEQ ID NO: 1309, SEQ ID NO: 1310, SEQ ID NO: 1311,respectively, and at both positions 11 and 19 for SEQ ID NO: 1312.

In certain embodiments of the invention, the Class 4 peptide comprisesthe amino acid sequence of any of SEQ ID NOs: 1362, 1364-1367, and 1371.

Modifications to Improve Solubility

The Class 4 peptides can be further modified to improve the peptide'ssolubility in aqueous solutions at physiological pH, while, in someaspects retaining a glucagon antagonist activity. Introduction ofhydrophilic groups at positions corresponding to positions 1, 16, 17,20, 21, 24 and 29 of the native peptide, or at the C-terminus, canimprove the solubility of the resulting Class 4 peptide in solutionshaving a physiological pH, while retaining the parent compoundsantagonist activity. Therefore, in some embodiments the presentlydisclosed Class 4 peptides are further modified to comprise one or morehydrophilic groups covalently linked to the side chains of amino acidscorresponding to amino acid positions 1, 16, 17, 20, 21, 24 and 29 ofthe native glucagon peptide or of the N- or C-terminal amino acid. In afurther embodiment the side chains of amino acids corresponding to aminoacid positions 16 and 24 of the native glucagon peptide are covalentlybound to hydrophilic groups, and in some embodiments the hydrophilicgroup is polyethylene glycol (PEG).

Applicants have also discovered that native glucagon can be modified byintroducing charge at its carboxy terminus to enhance the solubility ofthe peptide while retaining the agonist properties of the peptide. Theenhanced solubility allows for the preparation and storage of glucagonsolutions at near neutral pH. Formulating glucagon solutions atrelatively neutral pHs (e.g. pH of about 6.0 to about 8.0) improves thelong term stability of the Class 4 peptides.

Again, applicants anticipate that the Class 4 peptides disclosed hereincan be similarly modified to enhance their solubility in aqueoussolutions at relatively neutral pH (e.g. pH of about 6.0 to about 8.0)while retaining the antagonist properties of the parent protein.Accordingly, some embodiments of the present invention is directed to aClass 4 peptide of SEQ ID NO: 1339 that has been further modifiedrelative to the native amino acids present at positions 6-29 of the wildtype glucagon (SEQ ID NO: 1301) to add charge to the peptide by thesubstitution of native non-charged amino acids with charged amino acids,or the addition of charged amino acids to the carboxy terminus. Inaccordance with some embodiments, one to three of the non-charged nativeamino acids of the Class 4 peptide of SEQ ID NO: 1339 are replaced witha charged amino acid. In some embodiments the charged amino acid isselected from the group consisting of lysine, arginine, histidine,aspartic acid and glutamic acid. More particularly, applicants havediscovered that substituting the normally occurring amino acid atcorresponding position 28 and/or 29 relative to native glucagon withcharged amino acids, and/or the addition of one to two charged aminoacids at the carboxy terminus of the Class 4 peptide, enhances thesolubility and stability of the Class 4 peptides in aqueous solutions atphysiologically relevant pHs (i.e., a pH of about 6.5 to about 7.5).Accordingly, such modifications of the Class 4 peptide disclosed hereinare anticipated to have a similar effect on the solubility in aqueoussolutions, particularly at a pH ranging from about 5.5 to about 8.0,while retaining the parent peptide's biological activity.

In accordance with some embodiments the Class 4 peptide of SEQ ID NO:1339 is modified by the substitution of the native amino acid atcorresponding position 28 and/or 29 relative to native glucagon with anegatively charged amino acid (e.g., aspartic acid or glutamic acid) andoptionally the addition of a negatively charged amino acid (e.g.,aspartic acid or glutamic acid) to the carboxy terminus of the peptide.In an alternative embodiment the Class 4 peptide of SEQ ID NO: 1339 ismodified by the substitution of the native amino acid at correspondingposition 29 relative to native glucagon with a positively charged aminoacid (e.g., lysine, arginine or histidine) and optionally the additionof one or two positively charged amino acid (e.g., lysine, arginine orhistidine) on the carboxy terminus of the peptide. In accordance withsome embodiments a Class 4 peptide having improved solubility andstability is provided wherein the peptide comprises the amino acidsequence of SEQ ID NO: 1341 with the proviso that at least one aminoacids at position, 23, or 24 of SEQ ID NO: 1341 is substituted with anacidic amino acid, and/or an additional acidic amino acid is added atthe carboxy terminus of SEQ ID NO: 1341. In some embodiments the acidicamino acids are independently selected from the group consisting of Asp,Glu, cysteic acid and homocysteic acid.

In accordance with some embodiments a Class 4 peptide having improvedsolubility and stability is provided wherein the antagonist comprisesthe amino acid sequence of SEQ ID NO: 1341, SEQ ID NO: 1342, SEQ ID NO:1343 or SEQ ID NO: 1344, wherein at least one of the amino acids atpositions 23 or 24 is substituted with a non-native amino acid residue(i.e. at least one amino acid present at position 23 or 24 of the analogis an acidic amino acid different from the amino acid present at thecorresponding position in SEQ ID NO: 1307). In accordance with someembodiments a glucagon agonist is provided comprising the sequence ofSEQ ID NO: 1341 or 1342 with the proviso that when the amino acid atposition 23 is asparagine and the amino acid at position 24 isthreonine, the peptide further comprises one to two amino acids,independently selected from the group consisting of Lys, Arg, His, Aspor Glu, added to the carboxy terminus of the Class 4 peptide.

In another embodiment the solubility of the Class 4 peptide of SEQ IDNO: 1342 can be improved by covalently linking a hydrophilic moiety toan amino acid residue at position 11, 12, 15, 16, 19 or 24, and in someembodiments the hydrophilic moiety is linked to an amino acid atposition 11, 16 or 19, and in a further embodiment the hydrophilicmoiety is linked to amino acid 19. In some embodiments the hydrophilicmoiety is a plasma protein or the Fc portion of an immunoglobin, and inan alternative embodiment the hydrophilic moiety is a hydrophilichydrocarbon chain. In some embodiments the hydrophilic moiety ispolyethylene glycol, having a molecular weight selected from the rangeof about 1,000 to about 5,000 Daltons. In another embodiment thehydrophilic moiety is polyethylene glycol, having a molecular weight ofat least about 20,000 Daltons. In some embodiments the polyethylenemodified Class 4 peptide comprises the amino acids sequence of SEQ IDNO: 1309, SEQ ID NO: 1310, SEQ ID NO: 1311, SEQ ID NO: 1312, SEQ ID NO:1343, SEQ ID NO: 1344 or SEQ ID NO: 1345.

Modifications to Improve Stability

The Asp-Ser sequence at position 15-16 of native glucagon has beenidentified as a uniquely unstable dipeptide that leads to prematurechemical cleavage of the native hormone in aqueous buffers. For example,when maintained at 0.01N HCl at 37° C. for 2 weeks, more than 50% of thenative glucagon may be cleaved into fragments. The two liberatedcleavage peptides 1-15 and 16-29 are devoid of glucagon-like biologicalactivity and thus represent a limitation on the aqueous pre-formulationof glucagon and its related analogs. The selective chemical substitutionof the Asp at position 15 of the native glucagon peptide with Glu hasbeen observed to virtually eliminate chemical cleavage of the 15-16peptide bond.

Accordingly, it is expected that the Class 4 peptides of the presentinvention can be similarly modified to decrease their susceptibility topremature chemical cleavage in aqueous buffers. In accordance with someembodiments the Class 4 peptides described herein can be furthermodified to enhance their stability in aqueous solutions by replacingthe native aspartic amino acid, located at position 15 of the nativeglucagon peptide, with an amino acid selected from the group consistingof cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid.In accordance with some embodiments the aspartic acid residue atposition 10 of the Class 4 peptide of SEQ ID NO: 1339 can be substitutedwith an amino acid selected from the group consisting of cysteic acid,glutamic acid, homoglutamic acid and homocysteic acid, and in someembodiments the native aspartic acid at position 10 of SEQ ID NO: 1339is replaced with glutamic acid. In accordance with some embodiments aClass 4 peptide having improved stability in aqueous solutions isprovided wherein the antagonist comprises a sequence selected from thegroup consisting of SEQ ID NO: 1336, SEQ ID NO: 1340 and SEQ ID NO:1342. In a further embodiment the Class 4 peptide is amidated.

In accordance with some embodiments, increased stability by way ofreduced degradation of the Class 4 peptide described herein may also beachieved by substitution of the serine at position 16 (according to thenumbering of native glucagon) with glutamic acid, cysteic acid,homo-glutamic acid, or homo-cysteic acid. In a specific embodiment, theserine at position 16 (according to the native glucagon sequencenumbering) is replaced with glutamic acid. In a more specific aspect,the Class 4 peptide comprising such a modification comprises aC-terminal carboxylate and is not amidated.

In accordance with some embodiments, a Class 4 peptide is providedcomprising a glucagon peptide selected from the group consisting of SEQID NO: 1307, SEQ ID NO: 1336, SEQ ID NO: 1339, SEQ ID NO: 1340, SEQ IDNO: 1341, SEQ ID NO: 1342, SEQ ID NO: 1343 and SEQ ID NO: 1344, furthermodified by one or more additional amino acid substitutions at positionscorresponding to positions 11, 12, 15, 16, 19 and/or 24 of the nativeglucagon peptide, wherein the amino acid substitutions comprise asubstitution with an amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG. Thepeptide can be substituted with a naturally occurring amino acid or asynthetic (non-naturally occurring) amino acid. Synthetic ornon-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein. In some embodiments aClass 4 peptide is provided wherein the peptide comprises the sequenceof SEQ ID NO: 1307, SEQ ID NO: 1336, SEQ ID NO: 1339, SEQ ID NO: 1340,SEQ ID NO: 1341, SEQ ID NO: 1342, SEQ ID NO: 1343 and SEQ ID NO: 1344,and further comprises a polyethylene glycol chain bound to correspondingposition 21 or 24 of the native glucagon peptide. In a furtherembodiment the C-terminus of the Class 4 peptide is modified to replacethe carboxylic acid group with an amide group.

Fusion Peptides and Conjugates

The present disclosure also encompasses Class 4 peptide fusion peptideswherein a second peptide has been fused to the C-terminus of the Class 4peptide. More particularly, the fusion peptide may comprise a Class 4peptide peptide of SEQ ID NO: 1344 that further comprises an amino acidsequence of SEQ ID NO: 1319 (GPSSGAPPPS), SEQ ID NO: 1320 (Lys Arg AsnArg Asn Asn Ile Ala) or SEQ ID NO: 1321 (Lys Arg Asn Arg) linked to thec-terminal amino acid of the Class 4 peptide. In some embodiments theamino acid sequence of SEQ ID NO: 1319 (GPSSGAPPPS) is bound to aminoacid 24 of the Class 4 peptide of SEQ ID NO: 1342 through a peptidebond. In another embodiment the fusion peptide comprises a Class 4peptide peptide of SEQ ID NO: 1307, SEQ ID NO: 1336, SEQ ID NO: 1339,SEQ ID NO: 1340, SEQ ID NO: 1341 or SEQ ID NO: 1343 that furthercomprises an amino acid sequence of SEQ ID NO: 1319 (GPSSGAPPPS) linkedto amino acid 24 of the Class 4 peptide. In another embodiment thefusion peptide comprises a Class 4 peptide peptide of SEQ ID NO: 1307,SEQ ID NO: 1336, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1339, SEQID NO: 1341 or SEQ ID NO: 1343 that further comprises an amino acidsequence of SEQ ID NO: 1320, SEQ ID NO: 1321 or SEQ ID NO: 1353 linkedto amino acid 24 of the Class 4 peptide. In some embodiments the Class 4peptide fusion peptide comprises a sequence selected from the groupconsisting of SEQ ID NO: 1346 and SEQ ID NO 1347. In a furtherembodiment the C-terminus of the fusion peptide is modified to replacethe carboxylic acid group with an amide group.

In some embodiments a Class 4 peptide fusion peptide is provided whereinthe Class 4 peptide portion of the fusion peptide is selected from thegroup consisting of SEQ ID NO: 1303, SEQ ID NO: 1304, SEQ ID NO: 1305,SEQ ID NO: 1306, SEQ ID NO: 1307, SEQ ID NO: 1308, SEQ ID NO: 1309, SEQID NO: 1311, SEQ ID NO: 1312, SEQ ID NO: 1313, SEQ ID NO: 1314, SEQ IDNO: 1315, SEQ ID NO: 1310, SEQ ID NO: 1316, SEQ ID NO: 1317, SEQ ID NO:1318 and SEQ ID NO: 1339 and the sequence of SEQ ID NO: 1319 is fused tothe carboxy terminus of the Class 4 peptide portion, and wherein the PEGchain, when present, is selected from the range of 500 to 40,000Daltons. More particularly, in some embodiments the Class 4 peptidesegment is selected from the group consisting of SEQ ID NO: 1313, SEQ IDNO: 1314, SEQ ID NO: 1315, SEQ ID NO: 1316, SEQ ID NO: 1346 and SEQ IDNO: 1347 wherein the PEG chain is selected from the range of about 500to about 5,000 Daltons, and more particularly, in some embodiments thePEG chain is about 1,000 Daltons. In a further embodiment the C-terminusis modified to replace the carboxylic acid group with an amide group.

The Class 4 peptide may further comprise one to two charged amino acidsadded to the carboxy terminus. In some embodiments, wherein one to twocharged amino acids are added to the carboxy terminus of SEQ ID NO:1344, the amino acids are negatively charged amino acids, including forexample glutamic acid and aspartic acid. In some embodiments, the Class4 peptide comprises the sequence of SEQ ID NO: 1342 wherein at least oneof corresponding positions 27 and 28 relative to the native glucagonpeptide comprises an amino acid selected from the group consisting ofaspartic acid and glutamic acid and wherein SEQ ID NO: 1342 isoptionally modified to include an addition one to two negatively chargedamino acids added to the carboxy terminus. In some embodiments thenegatively charged amino acids are glutamic acid or aspartic acid.

The Class 4 peptides disclosed herein can be combined with other activeagents, including for example, insulin, to treat diseases or conditionsthat are characterized by excessive glucagon activity. In someembodiments, Class 4 peptides that have been modified to be covalentlybound to a PEG chain having a molecular weight of greater than 10,000Daltons can be administered in conjunction with insulin to help tomaintain stable blood glucose levels in diabetics. The Class 4 peptidesof the present disclosure can be co-administered with insulin as asingle composition, simultaneously administered as separate solutions,or alternatively, the insulin and the Class 4 peptide can beadministered at different times relative to one another. In someembodiments the composition comprising insulin and the compositioncomprising the Class 4 peptide are administered within 12 hours of oneanother. The exact ratio of the Class 4 peptide relative to theadministered insulin will be dependent in part on determining theglucagon levels of the patient, and can be determined through routineexperimentation.

Dimer Peptides

The present disclosure also encompasses multimers of the modified Class4 peptides disclosed herein. Two or more of the modified Class 4peptides can be linked together using standard linking agents andprocedures known to those skilled in the art. For example, dimers can beformed between two modified Class 4 peptides through the use ofbifunctional thiol crosslinkers and bi-functional amine crosslinkers,particularly for Class 4 peptides that have been substituted (atpositions 11, 16 or 19, for example) with cysteine, lysine ornithine,homocysteine or acetyl phenylalanine residues (e.g. SEQ ID NO: 1309, SEQID NO: 1310, SEQ ID NO: 1311 and SEQ ID NO: 1312). The dimer can be ahomodimer or alternatively can be a heterodimer. In some embodiments thedimer is formed between two Class 4 peptides independently selected fromthe group consisting of SEQ ID NO: 1308, SEQ ID NO: 1309, SEQ ID NO:1310, SEQ ID NO: 1311, SEQ ID NO: 1312, SEQ ID NO: 1345, SEQ ID NO:1346, or SEQ ID NO: 1347, wherein the two peptides are linked to oneanother via a linker attached to position 11 of each peptide, 16 of eachpeptide, or position 19 of each peptide or any combination thereof. Insome embodiments the linkage is a disulfide linkage between a Cys11 toCys11 or a Cys19 to Cys19 or a Cys11 to Cys19 residue of the respectiveClass 4 peptide peptides.

Similarly, a dimer can be formed between two Class 4 peptide peptidesindependently selected form the group consisting of SEQ ID NO: 1303, SEQID NO: 1304, SEQ ID NO: 1305, SEQ ID NO: 1306, SEQ ID NO: 1307, SEQ IDNO: 1308, SEQ ID NO: 1309, SEQ ID NO: 1310, SEQ ID NO: 1311, SEQ ID NO:1312, SEQ ID NO: 1336, SEQ ID NO: 1337, SEQ ID NO: 1338, SEQ ID NO: 1339and SEQ ID NO: 1342 wherein the linkage is formed between amino acidpositions independently selected from positions 16, 21 and 24 withrespect to the native glucagon peptide.

In accordance with some embodiments a Class 4 peptide dimer is providedcomprising two Class 4 peptides, each comprising the sequence of SEQ IDNO: 1346, wherein the two antagonists are linked to one another by adisulfide bond through amino acid position 25. In another embodiment aClass 4 peptide dimer is provided comprising two Class 4 peptides, eachcomprising the sequence of SEQ ID NO: 1347, wherein the two antagonistsare linked to one another by a disulfide bond through amino acidposition 35. In some embodiments the dimer is formed from Class 4peptides of SEQ ID NO: 1346 and SEQ ID NO: 1347 wherein the amino acidat position 10 is glutamic acid.

In some embodiments the dimer comprises a homodimer of a Class 4 peptidefusion peptide selected from the group consisting of SEQ ID NO: 1307,SEQ ID NO: 1308, SEQ ID NO: 1336, SEQ ID NO: 1337, SEQ ID NO: 1340, SEQID NO: 1339, NO: 1340, SEQ ID NO: 1341, SEQ ID NO: 1342 andpharmaceutically acceptable salts of said Class 4 peptides. Inaccordance with some embodiments a dimer is provided comprising a firstClass 4 peptide bound to a second Class 4 peptide via a linker, whereinthe first and second peptides of the dimer are independently selectedfrom the group consisting of SEQ ID NO: 1307, SEQ ID NO: 1308, SEQ IDNO: 1336, SEQ ID NO: 1337, SEQ ID NO: 1339, SEQ ID NO: 1340, SEQ ID NO:1341, and SEQ ID NO: 1342, and pharmaceutically acceptable salts of saidglucagon polypeptides. In another embodiment the first and second Class4 peptides of the dimer are independently selected from the groupconsisting of SEQ ID NO: 1307, SEQ ID NO: 1308, SEQ ID NO: 1336 and SEQID NO: 1339.

In another embodiment the dimer comprises a homodimer of a Class 4peptide selected from the group consisting of SEQ ID NO: 1323, SEQ IDNO: 1324, SEQ ID NO: 1325, SEQ ID NO: 1326, SEQ ID NO: 1327, SEQ ID NO:1328, SEQ ID NO: 1329, SEQ ID NO: 1330, SEQ ID NO: 1331. In anotherembodiment, a Class 4 peptide dimer is provided wherein the first andsecond peptides of the dimer comprise an amino acid sequenceindependently selected from the group consisting of SEQ ID NO: 1323, SEQID NO: 1324, SEQ ID NO: 1325, SEQ ID NO: 1326, SEQ ID NO: 1327 and SEQID NO: 1328. In another embodiment the dimer comprises a homodimer of aClass 4 peptide selected from the group consisting of SEQ ID NO: 1309,SEQ ID NO: 1311 and SEQ ID NO: 1312, wherein the peptide furthercomprises a polyethylene glycol chain covalently bound to position 11 or19 of the glucagon peptide.

The Class 4 glucagon related peptide may comprise the amino acidsequence of any of SEQ ID NOs: 1301-1371, optionally with up to 1, 2, 3,4, or 5 further modifications that retain glucagon antagonist activity.

Class 5 Glucagon Related Peptides

In certain embodiments, a glucagon related peptide is a class 5 glucagonrelated peptide (see, e.g., International (PCT) Patent ApplicationPublication No. WO 2009/058734, incorporated herein by reference in itsentirety).

All biological sequences referenced in the following section (SEQ IDNOs: 1401-1518) correspond to SEQ ID NOs.: 1-118 in International PatentApplication Publication No. WO 2009/058734.

Activity

In certain aspects a class 5 glucagon related peptide (hereafterreferred to as a “class 5 peptide”) may be a glucagon antagonist/GLP-1agonist. Glucagon antagonists/GLP-1 agonists are utilized in any settingwhere the suppression of glucagon agonism is desired while simultaneousstimulation of GLP-1 activity is also desired. For example, glucagonantagonist activity in conjunction with GLP-1 stimulation can be used inthe treatment of diabetes where glucagon antagonism has beendemonstrated in pre-clinical models of hyperglycemia to yield a loweringof blood glucose and GLP-1 activity is associated with insulinproduction. Compounds demonstrating GLP-1 activity have also been knownto be useful for treating obesity and preventing weight gain.

In certain aspects class 5 peptides are believed to be suitable for anyuse that has previously been described for other glucagonantagonist/GLP-1 agonists. These two activities have separately beenshown to be highly desirable properties for the treatment of themetabolic syndrome, specifically diabetes and obesity. The glucagonantagonist activity is useful in any setting where the suppression ofglucagon agonism is desired. The presence of GLP-1 agonism furthersuppresses the endogenous secretion of glucagon from the pancreas whilestimulating insulin synthesis and secretion. The two pharmacologicalactions serve in a synergistic fashion to normalize metabolicabnormalities. Accordingly, the Class 5 peptides can be used to treathyperglycemia, or treat other metabolic diseases that result from highblood levels of glucagon or high blood glucose levels. In accordancewith some embodiments the patient to be treated using the glucagonantagonist/GLP-1 agonists such as class 5 peptides disclosed herein is adomesticated animal, and in another embodiment the patient to be treatedis a human. Studies suggest that lack of glucagon suppression indiabetic patients contributes to postprandial hyperglycemia in part viaaccelerated glycogenolysis. Analysis of blood glucose during an OralGlucose Tolerance Test (OGTT), and in the presence or absence ofsomatostatin-induced glucagon suppression has shown a significantincrease in glucose in subjects with higher glucagon levels.Accordingly, a glucagon antagonist/GLP-1 agonists or Class 5 peptidesdescribed herein can be used to treating hyperglycemia, and are expectedto be useful for treating a variety of types of diabetes includingdiabetes mellitus type I, diabetes mellitus type II, or gestationaldiabetes, either insulin-dependent or non-insulin-dependent, andreducing complications of diabetes including nephropathy, retinopathyand vascular disease.

Such methods for reducing appetite or promoting loss of body weight areexpected to be useful in reducing body weight, preventing weight gain,or treating obesity of various causes, including drug-induced obesity,and reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases.

Pharmaceutical compositions comprising class 5 peptides can beformulated and administered to patients using standard pharmaucticallyacceptable carriers and routes of administration known to those skilledin the art. Accordingly, the present disclosure also encompassespharmaceutical compositions comprising one or more class 5 peptidesdisclosed herein in combination with a pharmaceutically acceptablecarrier. The pharmaceutical compositions may comprise the class 5peptides as the sole pharmaceutically active component, or the class 5peptides can be combined with one or more additional active agents. Inaccordance with some embodiments a composition is provided comprising aClass 5 peptide and insulin or an insulin analog. Alternatively, acomposition is provided for inducing weight loss or preventing weightgain can be provided that comprises the sequence of SEQ ID NO: 1415 orSEQ ID NO: 1451 further comprising the amino acid sequence of SEQ ID NO:1421 (GPSSGAPPPS) or SEQ ID NO: 1450 linked to amino acid 24 of SEQ IDNO: 1415 or SEQ ID NO: 1451, and an anti-obesity peptide. Suitableanti-obesity peptides include those disclosed in U.S. Pat. Nos.5,691,309, 6,436,435 or US Patent application 20050176643.

Class 5 Peptide Structure

In accordance with some embodiments a Class 5 peptide is providedcomprising a glucagon peptide that has been modified by the deletion ofthe first 1 to 5 amino acids residues (e.g., first amino acid, first twoamino acids, first three amino acids, first four amino acids, first fiveamino acids) from the N-terminus, and stabilization of the alpha-helixstructure in the C-terminal portion of the compound (around amino acidpositions 12-29 according to the amino acid numbering of wild typeglucagon, SEQ ID NO: 1401), e.g., by the linkage of the side chains ofamino acid pairs, selected from positions 12 and 16, 16 and 20, 20 and24, and 24 and 28 (relative to the native glucagon peptide sequence), toone another through hydrogen-bonding or ionic interactions, such as theformation of salt bridges, or by covalent bonds. Alternatively,stabilization of the alpha-helix around residues 12-29 is achievedthrough introduction of one or more α,α-disubstituted amino acids atpositions that retain the desired activity. In some embodiments, one,two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29(according to the amino acid numbering of wild type glucagon) of theClass 5 peptide or analog thereof is substituted with anα,α-disubstituted amino acid. For example, substitution of position 16(according to the amino acid numbering of wild type glucagon) of a Class5 peptide or analog thereof with amino iso-butyric acid (Aib) provides astabilized alpha helix in the absence of a salt bridge or lactam. Insome embodiments, one, two, three or more of positions 16, 20, 21 or 24(according to the amino acid numbering of wild type glucagon) aresubstituted with Aib.

In accordance with some embodiments, a class 5 peptide is providedwherein the peptide exhibits at least 80% of the maximum agonismachieved by native GLP-1 at the GLP-1 receptor, and exhibits glucagonantagonist activity that reduces the maximum glucagon-induced cAMPproduction at the glucagon receptor by at least about 50%, as measuredby cAMP production in an in vitro assay. In some embodiments, the class5 peptide exhibits at least 90% of the activity of native GLP-1 at theGLP-1 receptor, and exhibits glucagon antagonist activity, that reducesthe maximum glucagon-induced cAMP production at the glucagon receptor byat least about 80%.

In accordance with some embodiments the class 5 peptide comprises aderivative peptide of SEQ ID NO: 1402 wherein the peptide comprisesfurther amino acid substitutions relative to SEQ ID NO: 1402 at one tothree amino acid positions selected from positions 1, 2, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 19, 22 and 24, and exhibits at least 90%of the activity of native GLP-1 at the GLP-1 receptor, and exhibitsglucagon antagonist activity, that reduces the maximum glucagon-inducedcAMP production at the glucagon receptor by at least about 80%.

In some embodiments, the alpha-helix structure in the C-terminal portionof the Class 5 peptide (around amino acids 12-29 according to the aminoacid numbering of wild type glucagon) is stabilized by, e.g., formationof a covalent or non-covalent intramolecular bridge, or substitutionand/or insertion of amino acids around positions 12-29 with an alphahelix-stabilizing amino acid (e.g., an α,α-disubstituted amino acid). Insome embodiments, one, two, three, four or more of positions 16, 17, 18,19, 20, 21, 24 or 29 (according to the amino acid numbering of wild typeglucagon) of the Class 5 peptide or analog thereof is substituted withan α,α-disubstituted amino acid e.g., amino isobutyric acid (Aib). Forexample, substitution of position 16 (according to the amino acidnumbering of wild type glucagon) of a Class 5 peptide or analog thereofwith amino iso-butyric acid (Aib) provides a stabilized alpha helix inthe absence of a salt bridge or lactam.

In some embodiments the class 5 peptide comprises SEQ ID NO: 1415 or SEQID NO: 1451, and more particularly, a sequence selected from the groupconsisting of SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1407, SEQ IDNO: 1408, SEQ ID NO: 1409, SEQ ID NO: 1416, SEQ ID NO: 1417, SEQ ID NO:1418, SEQ ID NO: 1419, SEQ ID NO: 1422, SEQ ID NO: 1423, SEQ ID NO: 1424and SEQ ID NO: 1425. In further embodiments the class 5 peptidecomprises a derivative peptide of SEQ ID NO: 1415 or SEQ ID NO: 1451wherein the peptide comprises a further amino acid substitution relativeto SEQ ID NO: 1415 or SEQ ID NO: 1451 at one to three amino acidpositions selected from positions 1, 2, 5, 6, 8, 9, 12, 13 and 14. Insome embodiments the substitutions at positions 1, 2, 5, 6, 8, 9, 12, 13and 14 are conservative amino acid substitutions. In some embodimentsthe threonine at position 24 of SEQ ID NO: 1405 or SEQ ID NO: 1406 issubstituted with glycine.

In accordance with some embodiments the class 5 peptide represents afurther modification of the peptide wherein in addition to theN-terminal deletion, the phenylalanine at position 6 of the nativeglucagon peptide is modified, e.g., to comprise a hydroxyl group inplace of the N-terminus amino group. In a further embodiment the naturalcarboxylic acid of the C-terminal amino acid is replaced with acharge-neutral group, such as an amide or ester.

In accordance with some embodiments, Class 5 peptides have been preparedwherein the first three to five amino acids of native glucagon have beendeleted, the amino acid at position 9, relative to the native glucagonpeptide, has been substituted with an amino acid selected from the groupconsisting of glutamic acid, homoglutamic acid, β-homoglutamic acid, asulfonic acid derivative of cysteine, or an alkylcarboxylate derivativeof cysteine having the structure of:

wherein X₅ is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl, and thealpha-helix structure in the C-terminal portion of glucagon (aroundamino acids 12-29 according to the amino acid numbering of wild typeglucagon) is stabilized, e.g., via a lactam bridge is formed between theside chains of amino acids 12 and 16 or between amino acids 16 and 20,relative to the native glucagon peptide. Examples of amino acid pairingsthat are capable of covalently bonding to form a seven-atom linkingbridge are detailed through-out this disclosure. In some embodiments,the sulfonic acid derivative of cysteine is cysteic acid or homocysteicacid.

In some embodiments a class 5 is provided comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1405, SEQ IDNO: 1406, SEQ ID NO: 1407, or SEQ ID NO: 1408, wherein said peptidecomprises a lactam ring formed between the side chains of amino acids 7and 11 for SEQ ID NO: 1405, between 11 and 15 for SEQ ID NO: 1406,between positions 15 and 19 for SEQ ID NO: 1407 and between positions 19and 24 for SEQ ID NO: 1408, each of said sequences being furthermodified to comprise a hydrophilic moiety covalently bound to thepeptide. More particularly, in some embodiments each of the lactambearing class 5 peptide are modified by covalent attachment of apolyethylene glycol chain. For example, for a class 5 peptide comprisingSEQ ID NO: 1405, the peptide is pegylated at a position selected fromthe group consisting of 12, 15, 16, 19 and 24; for a class 5 peptidecomprising SEQ ID NO: 1406, the peptide is pegylated at a positionselected from the group consisting of 12, 16, 19 and 24; for a class 5peptide comprising SEQ ID NO: 1407, the peptide is pegylated at aposition selected from the group consisting of 11, 12, 16 and 24; forclass 5 peptide comprising SEQ ID NO: 1408, the peptide is pegylated ata position selected from the group consisting of 11, 12, 15 and 16. Inaccordance with some embodiments a class 5 peptide comprising SEQ ID NO:1447 or SEQ ID NO: 1448 is provided wherein the petide is pegylated at aposition selected from the group consisting of 12, 16, 19 and 24,relative to the SEQ ID NO: 1447 or SEQ ID NO: 1448 sequence. In afurther embodiment the peptide of SEQ ID NO: 1447 or SEQ ID NO: 1448 isfurther modified by the addition of the sequence of SEQ ID NO: 1421 tothe carboxy terminus of the peptide.

As detailed above in certain aspects Class 5 peptides are providedwherein the first five amino acids of native glucagon have been deleted,the amino group of the N-terminal amino acid (phenylalanine) has beenreplaced with a hydroxyl group (i.e., the first amino acid isphenyl-lactic acid) and the side chains of one or more amino acid pairsselected from positions 12 and 16, 16 and 20, 20 and 24, and 24 and 28are linked to one another, thus stabilizing the Class 5 peptide alphahelix.

In accordance with some embodiments a class 5 peptide is providedcomprising the sequence of SEQ ID NO: 1402 that is modified by asubstitution of the serine residue at position 11 of SEQ ID NO: 1402(position 16 according to the amino acid numbering of native glucagon)with an amino acid selected from the group consisting of glutamic acid,glutamine, homoglutamic acid, homocysteic acid, threonine or glycine. Inaccordance with some embodiments the serine residue at position 11 ofSEQ ID NO: 1402 is substituted with an amino acid selected from thegroup consisting of glutamic acid, glutamine, homoglutamic acid andhomocysteic acid, and in some embodiments the serine residue issubstituted with glutamic acid. In accordance with some embodiments theclass 5 peptide comprises the sequence of SEQ ID NO: 1438.

In some embodiments a class 5 peptide is provided wherein anintramolecular bridge is formed between two amino acid side chains tostabilize the three dimensional structure of the carboxy terminus of thepeptide of SEQ ID NO: 1402. More particularly, the side chains of one ormore amino acids selected from amino acid pairs 7 and 11, 11 and 15, 15and 19 or 19 and 23 of SEQ ID NO: 1402 are linked to one another, thusstabilizing the alpha helix in the C-terminal portion. The two sidechains can be linked to one another through hydrogen-bonding, ionicinteractions (such as the formation of salt bridges), or by covalentbonds. In accordance with some embodiments the size of the linker is 7-9atoms, and in some embodiments the size of the linker is 8 atoms. Insome embodiments the class 5 peptide is selected from the groupconsisting of SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1407 and SEQID NO: 1408. In some embodiments the C-terminal amino acid of the class5 peptide have an amide group substituting for the carboxylic acid groupthat is present on the native amino acid.

In accordance with some embodiments class 5 peptide is provided whereinthe analog comprises an amino acid sequence of SEQ ID NO: 1409. In someembodiments the three dimensional structure of the carboxy terminus ofthe peptide of SEQ ID NO: 1409 is stabilized by the formation ofcovalent bonds between the side chains of the peptide. In someembodiments two amino acid side chains are bound to one another to forma lactam ring. The size of the lactam ring can vary depending on thelength of the amino acid side chains, and in some embodiments the lactamis formed by linking the side chains of a lysine amino acid to aglutamic acid side chain. In some embodiments the C-terminal amino acidof the class 5 peptides have an amide group substituting for thecarboxylic acid group that is present on the native amino acid.

The order of the amide bond in the lactam ring can be reversed (e.g., alactam ring can be formed between the side chains of a Lys12 and a Glu16 or alternatively between a Glu 12 and a Lys16). In accordance withsome embodiments a glucagon analog of SEQ ID NO: 1409 is providedwherein at least one lactam ring is formed between the side chains of anamino acid pair selected from the group consisting of amino acid pairs 7and 11, 11 and 15, 15 and 19 or 19 and 23 of SEQ ID NO: 1409. In someembodiments a class 5 peptide is provided wherein the peptide comprisesthe sequence of SEQ ID NO: 1410, said sequence further comprising anintramolecular lactam bridge formed between amino acid positions 7 and11, or between amino acid positions 11 and 15, or between amino acidpositions 15 and 19 of SEQ ID NO: 1410. In some embodiments a class 5peptide is provided wherein the peptide comprises the sequence of SEQ IDNO: 1411, said sequence further comprising an intramolecular lactambridge formed between amino acid positions 7 and 11, or between aminoacid positions 11 and 15 of SEQ ID NO: 1411. In some embodiments theclass 5 peptide comprises the sequence of SEQ ID NO: 1417.

Additional class 5 peptide are provided comprising derivatives of SEQ IDNO: 1405, wherein the aspartic acid at position 10 of SEQ ID NO: 1405(position 15 of native glucagon) has been substituted with glutamicacid, an amino acid of the general structure:

wherein X₆ is C1-C3 alkyl, C2-C3 alkene or C2-C3 alkynyl, and in someembodiments X₆ is C1-C3 alkyl, and in another embodiment X₆ is C2 alkyl.In some embodiments a Class 5 peptide derivative of SEQ ID NO: 1409 isprovided wherein position 10 of SEQ ID NO: 1409 (position 15 of nativeglucagon) is substituted with an amino acid selected from the groupconsisting of glutamic acid, cysteic acid, homocysteic acid andhomoglutamic acid. In a further embodiment position 10 of SEQ ID NO:1409 is substituted with an amino acid selected from the groupconsisting of cysteic acid or homocysteic acid. In some embodiments aClass 5 peptide derivative of SEQ ID NO: 1406, SEQ ID NO: 1407 or SEQ IDNO: 1408 is provided wherein position 10 of SEQ ID NO: 1406, SEQ ID NO:1407 or SEQ ID NO: 1408 is substituted with an amino acid selected fromthe group consisting of glutamic acid, cysteic acid, homocysteic acidand homoglutamic acid. In some embodiments the C-terminal amino acid ofa class 5 peptide have an amide group substituting for the carboxylicacid group that is present on the native amino acid.

In some embodiments an amino acid of class 5 peptide is substituted withat least one cysteine residue, wherein the side chain of the cysteineresidue is further modified with a thiol reactive reagent, including forexample, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl, andhaloacyl. These thiol reactive reagents may contain carboxy, keto,hydroxyl, and ether groups as well as other hydrophilic moieties such aspolyethylene glycol units. In an alternative embodiment, an amino acidof a class 5 peptide is substituted with lysine, and the side chain ofthe substituting lysine residue is further modified using amine reactivereagents such as active esters (succinimido, anhydride, etc) ofcarboxylic acids or aldehydes of hydrophilic moieties such aspolyethylene glycol. In accordance with some embodiments the lysineresidue corresponding to position 7 of the peptide of SEQ ID NO: 1405 issubstituted with arginine and a single lysine substitution is insertedfor one of the amino acids corresponding to position 12, 15, 16, 19 and24 of SEQ ID NO: 1405.

In another embodiment the methionine residue corresponding to position22 of the class 5 peptides disclosed herein is changed to leucine ornorleucine to prevent oxidative degradation of the peptide.

Moreover class 5 peptides, in some aspects, also encompass amino acidsubstitutions at positions that are known not to be critical to thefunction of the glucagon analog. In some embodiments the substitutionsare conservative amino acid substitutions at one, two or three positionsselected from the group consisting of 2, 5, 6, 7, 8, 9, 12, 13, 14, 15,16, 19, 22, 23 or 24. In some embodiments the amino acids correspondingto positions 16, 17, 20, 21, 24 or 29 of the native glucagon peptide,and more particularly at position 21 and/or 24 relative to nativeglucagon are substituted with cysteine or lysine, wherein a PEG chain iscovalently attached to the substituted cysteine or lysine residue.

In accordance with some embodiments, a class 5 peptide is providedcomprising a sequence consisting of SEQ ID NO: 1409, further modified byone or more additional amino acid substitutions at positionscorresponding to positions 11, 12, 15, 16, 19 and/or 24 of the peptide(including for example substitution with cysteine), wherein the aminoacid substitution comprises an amino acid having a side chain suitablefor crosslinking with hydrophilic moieties, including for example, PEG.Native glucagon can be substituted with a naturally occurring amino acidor a synthetic (non-naturally occurring) amino acid. Synthetic ornon-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein. In some embodiments aClass 5 peptide is provided wherein the peptide comprises the sequenceof SEQ ID NO: 1409 and further comprises a polyethylene glycol chainbound to position 16 or 19 of the peptide. In a further embodiment theC-terminus of the glucagon analog is modified to replace the carboxylicacid group with an amide group.

In accordance with some embodiments a class 5 peptide is providedcomprising a glucagon analog selected from the group consisting of:

(SEQ ID NO: 1439) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Xaa-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Asn- Thr-R₂(SEQ ID NO: 1413) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Gln-Trp-Leu-Xaa-Asn- Thr-R₂,(SEQ ID NO: 1414) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Xaa-Trp-Leu-Xaa-Asn- Thr-R₂ and(SEQ ID NO: 1412) R₁-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Xaa-Trp-Leu-Xaa-Asn- Thr-R₂,wherein Xaa at position 4=aspartic acid, glutamic acid, cysteic acid orhomocysteic acid, Xaa at position 10=Asp, Glu, cysteic acid,homoglutamic acid and homocysteic acid, Xaa at position 16 is Asp, Cys,Orn, homocysteine or acetyl phenylalanine and the Xaa at position 19 isGln, Cys, Orn, homocysteine and acetyl phenylalanine, Xaa at position22=Met, Leu or Nle, R₁ is OH or NH₂, and R₂ is Gly Pro Ser Ser Gly AlaPro Pro Pro Ser (SEQ ID NO: 1421), Gly Pro Ser Ser Gly Ala Pro Pro ProSer Xaa (SEQ ID NO: 1450; wherein Xaa is Cys, Orn, homocystein or acetylphenyalanine), COOH or CONH₂, wherein the peptide is optionallypegylated at position 16 of SEQ ID NO: 1413, position 19 of SEQ ID NO:1414 and at positions 16 and 19 of SEQ ID NO: 1412. In some embodimentsthe Thr at position 24 of SEQ ID NOs: 1412-1414 and 1439 is substitutedwith Gly. In accordance with some embodiments the peptide comprises thesequence of SEQ ID NO: 13 or SEQ ID NO: 1414, wherein R₁ is OH. Inaccordance with some embodiments the peptide comprises the sequence ofSEQ ID NO: 1413 or SEQ ID NO: 1414, wherein R₁ is OH and R₂ is CONH₂. Inaccordance with some embodiments the peptide comprises the sequence ofSEQ ID NO: 1413 or SEQ ID NO: 1414, wherein R₁ is OH, R2 is CONH₂ andthe threonine at position 24 is substituted with glycine.

In some embodiments, a class 5 peptide is further modified to compriseone or more amino acids of native GLP-1 by substitution of the nativeglucagon residue(s) at corresponding amino acid positions. For example,the class 5 peptide may comprise one or more amino acid substitutions atany of positions 2, 3, 17, 18, 21, 23, and 24 (according to the aminoacid numbering of native glucagon). In a specific embodiment, the class5 peptide is modified by one or more of the following amino acidsubstitutions: Ser2 is replaced with Ala, Gln3 is replaced with Glu,Arg17 is replaced with Gln, Arg at position 18 is replaced with Ala, Aspat position 21 is replaced with Glu, Val at position 23 is replaced withIle, and Gln at position 24 is replaced with Ala (amino acid positionsare in accordance with the native glucagon sequence). In a specificembodiment, the class 5 peptide is modified by replacing Ser2 with Alaand Gln3 with Glu (according to the amino acid numbering of nativeglucagon). In another specific embodiment, the class 5 peptide ismodified with all of the following amino acid substitutions: Arg17 isreplaced with Gln, Arg at position 18 is replaced with Ala, Asp atposition 21 is replaced with Glu, Val at position 23 is replaced withIle, and Gln at position 24 is replaced with Ala (amino acid numberingaccording to native glucagon). In yet another specific embodiment, theclass 5 peptide is modified to comprise just Glu at position 21(according to the numbering of SEQ ID NO: 1401). Accordingly, the class5 peptide can comprise the amino acid sequence of any of SEQ ID NOs:1460-1470, 1473-1478, 1480-1488, 1490-1496, 1503, 1504, 1506, and1514-1518.

Also provided herein is a class 5 peptide or conjugate thereofcomprising (1) a stabilized alpha helix through means described herein(e.g., through an intramolecular bridge, or incorporation of one or morealpha, alpha-di-substituted amino acids, or an acidic amino acid atposition 16 (according to the numbering of SEQ ID NO:1401), or acombination thereof; (2) a C-terminal amide or ester in place of aC-terminal carboxylate, and (3) a general structure of A-B-C,

wherein A is selected from the group consisting of

(i) phenyl lactic acid (PLA);

(ii) an oxy derivative of PLA; and

(iii) a peptide of 2 to 6 amino acids in which two consecutive aminoacids of the peptide are linked via an ester or ether bond;

wherein B represents amino acids p to 26 of SEQ ID NO: 1401, wherein pis 3, 4, 5, 6, or 7, optionally comprising one or more amino acidmodifications, as described herein, including, for example, any of themodifications described for Class 5 peptides. For instance the one ormore modification may be selected from the group consisting of:

(iv) Asp at position 9 (according to the amino acid numbering of SEQ IDNO: 1401) is substituted with a Glu, a sulfonic acid derivative of Cys,homoglutamic acid, (3-homoglutamic acid, or an alkylcarboxylatederivative of cysteine having the structure of:

wherein X₅ is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl;

(v) substitution of one or two amino acids at positions 10, 20, and 24,(according to the amino acid numbering of SEQ ID NO: 1401) with an aminoacid covalently attached to an acyl or alkyl group via an ester, ether,thioether, amide, or alkyl amine linkage;

(vi) substitution of one or two amino acids at positions 16, 17, 20, 21,and 24 (according to the amino acid numbering of SEQ ID NO: 1401) withan amino acid selected from the group consisting of: Cys, Lys,ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein theamino acid of the group is covalently attached to a hydrophilic moiety;

(vii) Asp at position 15 (according to the numbering of SEQ ID NO: 1401)is substituted with cysteic acid, glutamic acid, homoglutamic acid, andhomocysteic acid;

(viii) Ser at position 16 (according to the numbering of SEQ ID NO:1401) is substituted with cysteic acid, glutamic acid, homoglutamicacid, and homocysteic acid;

(ix) Arg at position 17 is replaced with Gln, Arg at position 18 isreplaced with Ala, Asp at position 21 is replaced with Glu, Val atposition 23 is replaced with Ile, and Gln at position 24 is replacedwith Ala (according to amino acid numbering of SEQ ID NO: 1401);

(x) Ser at position 16 is replaced with Glu, Gln at position 20 isreplaced with Glu, or Gln at position 24 is replaced with Glu (accordingto the amino acid numbering of SEQ ID NO: 1401);

wherein C (of the general structure of A-B-C) is selected from the groupconsisting of:

(vii) X;

(viii) X-Y;

(ix) X-Y-Z;

(x) X-Y-Z-R10;

wherein X is Met, Leu, or Nle; Y is Asn or a charged amino acid; Z isThr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine(Ac-Phe), or a charged amino acid; wherein R10 is selected from a groupconsisting of SEQ ID NOs: 1421, 1426, 1427, and 1450.

In a specific aspect, the peptide comprises an oxy derivative of PLA. Asused herein “oxy derivative of PLA” refers to a compound comprising amodified structure of PLA in which the hydroxyl group has been replacedwith O—R₁₁, wherein R₁₁ is a chemical moiety. In this regard, the oxyderivative of PLA can be, for example, an ester of PLA or an ether ofPLA.

Methods of making oxy derivatives of PLA are known in the art. Forexample, when the oxy derivative is an ester of PLA, the ester may beformed by upon reaction of the hydroxyl of PLA with a carbonyl bearing anucleophile. The nucleophile can be any suitable nucleophile, including,but not limited to an amine or hydroxyl. Accordingly, the ester of PLAcan comprise the structure of Formula IV:

wherein R₇ is an ester formed upon reaction of the hydroxyl of PLA witha carbonyl bearing a nucleophile.

The carbonyl bearing a nucleophile (which reacts with the hydroxyl ofPLA to form an ester) can be, for example, a carboxylic acid, acarboxylic acid derivative, or an activated ester of a carboxylic acid.The carboxylic acid derivative can be, but is not limited to, an acylchloride, an acid anhydride, an amide, an ester, or a nitrile. Theactivated ester of a carboxylic acid can be, for example,N-hydroxysuccinimide (NHS), tosylate (Tos), a carbodiimide, or ahexafluorophosphate. In some embodiments, the carbodiimide is1,3-dicyclohexylcarbodiimide (DCC), 1,1′-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or1,3-diisopropylcarbodiimide (DICD). In some embodiments, thehexafluorophosphate is selected from a group consisting ofhexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), ando-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU).

Methods of making ethers from reaction with a hydroxyl group (e.g., thehydroxyl of PLA) also are known in the art. For example, the hydroxylgroup of PLA may be reacted with a halogenated alkyl or tosylated alkylalcohol to form an ether bond.

In a specific embodiment, the chemical moiety bound to PLA via anoxygen-containing bond (e.g., via an ester or ether bond) is a polymer(e.g., a polyalkylene glycol), a carbohydrate, an amino acid, a peptide,or a lipid, e.g., a fatty acid or a steroid.

In a specific embodiment, the chemical moiety is an amino acid, which,optionally, is a part of a peptide, such that Formula IV is adepsipeptide. In this regard, PLA may be at a position other than theN-terminal amino acid residue of the peptide, such that the peptidecomprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) amino acidsN-terminal to the PLA residue. For example, the peptide can comprise PLAat position n, wherein n is 2, 3, 4, 5, or 6 of the peptide.

The amino acids N-terminal to the PLA residue may be synthetic ornaturally-occurring. In a specific embodiment, the amino acids which areN-terminal PLA are naturally-occurring amino acids. In some embodiments,the amino acids which are N-terminal to PLA are the N-terminal aminoacids of native glucagon. For example, the peptide can comprise at theN-terminus the amino acid sequence of any of SEQ ID NOs: 1452-1456,wherein PLA is linked to threonine via an ester bond:

His-Ser-Gln-Gly-Thr-PLA SEQ ID NO: 1452 Ser-Gln-Gly-Thr-PLASEQ ID NO: 1453 Gln-Gly-Thr-PLA SEQ ID NO: 1454 Gly-Thr-PLASEQ ID NO: 1455 Thr-PLA SEQ ID NO: 1456

In an alternative embodiment, one or more of the N-terminal amino acidsmay be substituted with an amino acid other than the amino acid ofnative glucagon. For example, when the peptide comprises PLA as theamino acid at position 5 or 6, the amino acid at position 1 and/orposition 2 may be an amino acid which reduces susceptibility to cleavageby dipeptidyl peptidase IV. More particularly, in some embodiments,position 1 of the peptide is an amino acid selected from the groupconsisting of D-histidine, alpha, alpha-dimethyl imidiazole acetic acid(DMIA), N-methyl histidine, alpha-methyl histidine, imidazole aceticacid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. More particularly, in some embodiments, position 2 ofthe antagonist/agonist peptide is an amino acid selected from the groupconsisting of D-serine, D-alanine, valine, glycine, N-methyl serine,N-methyl alanine, and aminoisobutyric acid (Aib). Also, for example,when the peptide comprises PLA as the amino acid at position 4, 5, or 6,the amino acid at position 3 of the peptide may be glutamic acid, asopposed to the native glutamine residue of native glucagon. In anexemplary embodiment of the invention, the peptide comprises at theN-terminus the amino acid sequence of any of SEQ ID NOs: 1457-1459.

With respect to the peptides comprising a compound of Formula IV, thepolymer may be any polymer, provided that it can react with the hydroxylgroup of PLA. The polymer may be one that naturally or normallycomprises a carbonyl bearing a nucleophile. Alternatively, the polymermay be one which was derivatized to comprise the carbonyl bearing thecarbonyl. The polymer may be a derivatized polymer of any of:polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

The polymer can be a biodegradable polymer, including a syntheticbiodegradable polymer (e.g., polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone)), and a naturalbiodegradable polymer (e.g., alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins (e.g., zein and other prolamines and hydrophobic proteins)), aswell as any copolymer or mixture thereof. In general, these materialsdegrade either by enzymatic hydrolysis or exposure to water in vivo, bysurface or bulk erosion.

The polymer can be a bioadhesive polymer, such as a bioerodible hydrogeldescribed by H. S. Sawhney, C. P. Pathak and J. A. Hubbell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer. Suitablewater-soluble polymers are known in the art and include, for example,polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel),hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose,hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose,hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose(Ethocel), hydroxyethyl cellulose, various alkyl celluloses andhydroxyalkyl celluloses, various cellulose ethers, cellulose acetate,carboxymethyl cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, vinyl acetate/crotonic acid copolymers,poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylicacid copolymers, polymethacrylic acid, polymethylmethacrylate, maleicanhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium andcalcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers,carboxypolymethylene, carboxyvinyl polymers, polyoxyethylenepolyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,carboxymethylamide, potassium methacrylate divinylbenzene co-polymer,polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, andcombinations thereof.

In a specific embodiment, the polymer is a polyalkylene glycol,including, for example, polyethylene glycol (PEG).

The carbohydrate may be any carbohydrate provided that it comprises oris made to comprise a carbonyl with an alpha leaving group. Thecarbohydrate, for example, may be one which has been derivatized tocomprise a carbonyl with an alpha leaving group. In this regard, thecarbohydrate may be a derivatized form of a monosaccharide (e.g.,glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose,maltose), an oligosaccharide (e.g., raffinose, stachyose), apolysaccharide (a starch, amylase, amylopectin, cellulose, chitin,callose, laminarin, xylan, mannan, fucoidan, galactomannan.

The lipid may be any lipid comprising a carbonyl with an alpha leavinggroup. The lipid, for example, may be one which is derivatized tocomprise the carbonyl. In this regard, the lipid, may be a derivative ofa fatty acid (e.g., a C4-C30 fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide. In some embodiments, the lipid is an oil, wax, cholesterol,sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride,or a phospholipid.

In some embodiments, R₇ has a molecular weight of about 100 kDa or less,e.g., about 90 kDa or less, about 80 kDa or less, about 70 kDa or less,about 60 kDa or less, about 50 kDa or less, about 40 kDa or less.Accordingly, R7 can have a molecular weight of about 35 kDa or less,about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about15 kDa or less, about 10 kDa or less, about 5 kDa or less, or about 1kDa.

In an alternative embodiment, the peptide comprising the generalstructure of A-B-C comprises, as A, a peptide of 2 to 6 amino acids inwhich two consecutive amino acids of the peptide of A are linked via anester or ether bond. The ester or ether bond may be, e.g., between aminoacids 2 and 3, 3 and 4, 4 and 5, or 5 and 6. Optionally the peptide of Amay be further modified by covalent linkage to another chemical moietyincluding linkage to a polymer (e.g. a hydrophilic polymer), alkylation,or acylation.

In a specific embodiment, the above-described class 5 peptide comprisingPLA is modified to comprise an oxy derivative of PLA, such as, forinstance, an ester of PLA or an ether of PLA. For example, the class 5peptide can comprise the amino acid sequence of any of SEQ ID NOs: 1402,1405-1420, 1422-1425, 1432-1436, 1438, 1439, 1445, 1446, and 1451,wherein the PLA is linked via an ester or ether bond to an amino acid,peptide, polymer, acyl group, or alkyl group. The amino acid, peptide,polymer, acyl group, or alkyl group may be any of those describedherein. In the case that the PLA is linked via an ester bond to an aminoacid or peptide, the class 5 peptide may be considered as adepsipeptide.

Also, in another specific embodiment, the above-described class 5peptide which lacks PLA is modified to comprise at least one ester bondor ether bond between two consecutive amino acids which are N-terminalto the amino acid at position 7 (according to the numbering of nativeglucagon). In a specific embodiment, the class 5 peptide comprises atleast one ester or ether bond between the two consecutive amino acids.In a more specific embodiment, the Class 5 peptide comprises theN-terminal 6 amino acids of SEQ ID NO: 1401 and two consecutive aminoacids of the N-terminal 6 amino acids are linked via an ester or etherbond.

The peptide of A may comprise any amino acids, synthetic or naturallyoccurring, provided that at least two consecutive amino acids are linkedvia an ester or ether bond. In a specific embodiment, the peptide of Acomprises amino acids of native glucagon. The amino acid at position 1and/or position 2 may be an amino acid which reduces susceptibility tocleavage by dipeptidyl peptidase IV. For instance, the peptide of A cancomprise at position 1 an amino acid selected from the group consistingof D-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA),N-methyl histidine, alpha-methyl histidine, imidazole acetic acid,desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. More particularly, in some embodiments, position 2 ofthe peptide of A is an amino acid selected from the group consisting ofD-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine,and aminoisobutyric acid (Aib). Also, for example, the amino acid atposition 3 of the peptide of A may be glutamic acid, as opposed to thenative glutamine residue of native glucagon. Accordingly, the peptide ofgeneral structure of A-B-C can comprise an amino acid sequence of:

Xaa1-Xaa2-Xaa3-Thr-Gly-Phe; (SEQ ID NO: 1507) Xaa2-Xaa3-Thr-Gly-Phe;(SEQ ID NO: 1508) or Xaa3-Thr-Gly-Phe; (SEQ ID NO: 1509)wherein Xaa1 is selected from a group consisting of: His, D-histidine,alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine,alpha-methyl histidine, imidazole acetic acid, desaminohistidine,hydroxyl-histidine, acetyl-histidine and homo-histidine; Xaa2 isselected from a group consisting of: Ser, D-serine, D-alanine, valine,glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid(Aib); and Xaa3 is Gln or Glu.

In some embodiments, B is modified by up to three amino acidmodifications. For example, B, which represents native amino acidsequence of SEQ ID NO: 1401 is modified by one or more conservativeamino acid modifications.

In another embodiment, B comprises one or more amino acid modificationsselected from the group consisting of (iv) to (x), as described herein.In a specific embodiment, B comprises one or both of the amino acidmodifications (v) and (vi). In a further specific embodiment, Bcomprises one or a combination of amino acid modifications selected fromthe group consisting of (iv), (vii), (viii), (ix), and (x), in additionto (v) and (vi).

As described herein, the peptide comprising the general structure A-B-Cmay comprise one or more charged amino acids at the C-terminus, e.g., asY and/or Z, as described herein. Alternatively or additionally, thepeptide comprising the general structure A-B-C may further comprise oneto two charged amino acids C-terminal to Z, when C comprises X-Y-Z. Thecharged amino acids can be, for example, one of Lys, Arg, His, Asp, andGlu. In a specific embodiment, Y is Asp.

In some embodiments, the peptide comprising the general structure A-B-Ccomprises a hydrophilic moiety covalently bound to an amino acid residueat position 1, 16, 20, 21, or 24 (according to the amino acid numberingof SEQ ID NO: 1401), or at the N- or C-terminal residue of the peptidecomprising the general structure A-B-C. In a specific embodiment, thehydrophilic moiety is attached to a Cys residue of the peptidecomprising the general structure A-B-C. In this regard, the amino acidat position 16, 21, 24, or 29 of native glucagon (SEQ ID NO: 1401) maybe substituted with a Cys residue. Alternatively, a Cys residuecomprising a hydrophilic moiety may be added to the C-terminus of thepeptide comprising the general structure A-B-C as position 30 or asposition 40, e.g., when the peptide comprising the general structureA-B-C comprises a C-terminal extension (positions according to the aminoacid numbering of SEQ ID NO: 1401). Alternatively, the hydrophilicmoiety may be attached to the PLA of the peptide comprising the generalstructure A-B-C via the hydroxyl moiety of PLA. The hydrophilic moietycan be any of those described herein, including, for example,polyethylene glycol.

In a specific aspect, the peptide comprising the general structure A-B-Ccomprises a stabilized alpha helix by virtue of incorporation of anintramolecular bridge. In some embodiments, the intramolecular bridge isa lactam bridge. The lactam bridge may be between the amino acids atpositions 9 and 12, the amino acids at positions 12 and 16, the aminoacids at positions 16 and 20, the amino acids at positions 20 and 24, orthe amino acids at positions 24 and 28 (according to the amino acidnumbering of SEQ ID NO: 1401). In a specific embodiment, the amino acidsat positions 12 and 16 or at positions 16 and 20 (according to the aminoacid numbering of SEQ ID NO: 1401) are linked via a lactam bridge. Otherpositions of the lactam bridge are contemplated.

Additionally or alternatively, the peptide comprising the generalstructure A-B-C can comprise an alpha, alpha di-substituted amino acidat, for example, any of positions 16, 20, 21, or 24 (according to theamino acid numbering of SEQ ID NO: 1401). In some embodiments, thealpha, alpha di-substituted amino acid is Aib. In a specific aspect, theAib is located at position 16 (according to the numbering of SEQ ID NO:1401).

Alternatively or additionally, the peptide comprising the generalstructure A-B-C may be modified to comprise an acidic amino acid atposition 16 (according to the numbering of SEQ ID NO: 1401), whichmodification enhances the stability of the alpha helix. The acidic aminoacid, in some embodiments, is an amino acid comprising a side chainsulfonic acid or a side chain carboxylic acid. In a more specificembodiment, the acidic amino acid is selected from the group consistingof Glu, Asp, homoglutamic acid, a sulfonic acid derivative of Cys,cysteic acid, homocysteic acid, Asp, and an alkylated derivative of Cyshaving the structure of

wherein X₅ is C1-C4 alkyl, C2-C4 alkenyl, or C2-C4 alkynyl.

In a specific embodiment, the Class 5 peptide may comprise the aminoacid sequence of any of SEQ ID NOs: 1460-1470, 1473-1478, 1480-1488,1490-1496, 1503, 1504, 1506, and 1514-1518, or comprising the amino acidsequence of any of Peptides 2-6 of Table 13, Peptides 1-8 of Table 14,and Peptides 2-6, 8, and 9 of Table 15.

TABLE 13 Lactam, Cex glucagon(6-39) peptides and glucagon antagonist and GLP-1 agonist activityGLP-1 EC_(so) Glu IC_(so) (nM) (nM) 1 E9, K12, E16FTSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 1451 762 (SEQ ID NO: 1428) 2E9, K12E16(lactam) FTSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 63 2008(SEQ ID NO: 1429) 3 E9, E16K20(lactam)FTSEYSKYLDERRAKDFVQWLMNTGPSSGAPPPS 36 42 (SEQ ID NO: 1430) 4D9, K12E16(Lactam) FTSDYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 118.7 828(SEQ ID NO: 1431) 5 [PLA6, E9, K12E16(Lactam)PLA-TSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 6 72 (SEQ ID NO: 1432) 6[PLA6, E9, E16K20(Lactam)] PLA-TSEYSKYLDERRAKDFVQWLMNTGPSSGAPPPS 20 20(SEQ ID NO: 1433)

TABLE 14  Lactam glucagon(1-29, 2-29, 4-29 and 6-29) peptides and theirglucagon antagonist and GLP-1 agonist activity Glucagon GLP-1 EC50 IC50(nM) (nM) Glucagon (SEQ ID NO: 1)  0.2~1.0*HSQGTFTSDYSKYLDSRRAQDFVQWLMNT GLP-1 (aa 1-30)(SEQ ID NO: 1404) 0.02~0.1HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR 1[PLA6, D9, E16K20(lactam), D28]G(6-29)(SEQ ID NO: 1460)   5~25 10~30 PLA TSDYSKYLDERRAKDFVQWLMDT 2[PLA6, D9, K12E16(Lactam), D28]G(6-29)(SEQ ID NO: 1675) 177 63PLA TSDYSKYLDERRAQDFVQWLMDT 3[PLA6, D9, E16, K20E24(Lactam), D28]G(6-29)(SEQ ID NO: 1477) 239 74PLA TSDYSKYLDERRAKDFVEWLMDT 4[PLA6, D9, E16, E24K28(lactam)]G(6~29)(SEQ ID NO: 1478) 289 22PLA TSDYSKYLDERRAQDFVEWLMKT 5[E9, E16K20(lactam), D28]G(4~29)(SEQ ID NO: 1485) 151 10~30 GTFTSEYSKYLDERRAKDFVQWLMDT 6[E9, E16K20(lactam), D28]G(2~29)(SEQ ID NO: 1486) 203  49 (PA)SQGTFTSEYSKYLDERRAKDFVQWLMDT 7[A2E3, E16K20(Lactam), D28]G(2~29)(SEQ ID NO: 1463) 175 63AEGTFTSEYSKYLDERRAKDFVQWLMDT 8[A2E3, E16K20(Lactam), D28]G(1~29)(SEQ ID NO: 1483) 0.2 130 (PA)HAEGTFTSEYSKYLDERRAKDFVQWLMDT 9 ANK2 (Bayer peptide)(SEQ ID NO: 1437)0.28 agonist HSQGTFTSDY ARYLDARRAREFIKWL VRGRG *EC50 at glucagonreceptor (PA = partial antagonist)

TABLE 15  Profile of Mixed Agonist/Antagonist Glucagon (6-CEX) Analogs 1E9, K12, E16 FTSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 1451 762(SEQ ID NO: 1428) 2 E9, K12E16(lactam)FTSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 63 2008 (SEQ ID NO: 1429) 3E9, E16K20(lactam) FTSEYSKYLDERRAKDFVQWLMNTGPSSGAPPPS 36 42(SEQ ID NO: 1430) 4 D9, K12E20(lactam)FTSDYSKYLDERRAQDFVQWLlMNTGPSSGAPPPS 18 828 (SEQ ID NO: 1431) 5[PLA6, E9, K12E20(lactam) PLA-TSEYSKYLDERRAQDFVQWLMNTGPSSGAPPPS 6 72(SEQ ID NO: 1432) 6 [PLA6, E9, E16K20(Lactam)]PLA-TSEYSKYLDERRAKDFVQWLMNTGPSSGAPPPS 20 20 (SEQ ID NO: 1433)Glucagon D⁹(6-29) analogs GLP-1 Glucagon EC50 (nM) IC50 (nM) 7PLA 6, D9, D28 PLA-TSDYSKYLDSRRAQDFVQWLMDT −700 tbd (SEQ ID NO: 1434) 8PLA6, D9, K12E20(Lactam) PLA-TSDYSKYLDERRAQDFVQWLMDT 21 13(SEQ ID NO: 1475) 9 PLA6, D9, E16K20(lactam) PLA-TSDYSKYLDERRAKDFVQWLMDT4 6 (SEQ ID NO: 1476)

In some embodiments, the peptide comprising the general structure A-B-Cis a Class 5 peptide. In a specific embodiment, the peptide exhibits atleast about 50% of the maximum agonism achieved by native GLP-1 at theGLP-1 receptor and at least about 50% inhibition of the maximum responseachieved by native glucagon at the glucagon receptor. In anotherspecific embodiment, the peptide exhibits at least about 55%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, or about 100% of the maximum agonism achieved bynative GLP-1 at the GLP-1 receptor. Alternatively or additionally, thepeptide may exhibit at least about 55%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,or about 100% inhibition of the maximum response achieved by nativeglucagon at the glucagon receptor.

In some embodiments, a peptide with Class 5 peptide or conjugatethereof, is provided comprising:

(1) modifications that confer glucagon antagonist activity, includingbut not limited to:

(a) substitution of the Phe at position 6 with PLA (according to aminoacid numbering of wild type glucagon), optionally with deletion of 1 to5 amino acids from the N-terminus of wild type glucagon; or

(b) deletion of 2 to 5 amino acids from the N-terminus of wild typeglucagon; optionally with substitution of Asp at position 9 of wild typeglucagon with glutamic acid, homoglutamic acid or a sulfonic acidderivative of cysteine (according to amino acid numbering of wild typeglucagon); and

(2) modifications that confer GLP-1 agonist activity, including but notlimited to:

(a) insertion or substitution of α,α-disubstituted amino acid withinamino acids 12-29 of wild type glucagon, e.g. at one, two, three, fouror more of positions 16, 17, 18, 19, 20, 21, 24 or 29 (according to theamino acid numbering of wild type glucagon); or

(b) introduction of an intramolecular bridge within amino acids 12-29 ofwild type glucagon, e.g. a salt bridge or a lactam bridge or anothertype of covalent bond; or

(c) substitution of the amino acid at one or more of positions 2, 3, 17,18, 21, 23, or 24 (according to the amino acid numbering of nativeglucagon) with the corresponding amino acid of GLP-1, e.g. Ser2 isreplaced with Ala, Gln3 is replaced with Glu, Arg17 is replaced withGln, Arg at position 18 is replaced with Ala, Asp at position 21 isreplaced with Glu, Val at position 23 is replaced with Ile, and/or Glnat position 24 is replaced with Ala; or

(d) other modifications that stabilize the alpha-helix structure aroundamino acid positions 12-29 according to the amino acid numbering of wildtype glucagon; and

(3) other modifications that enhance GLP-1 agonist activity, e.g. aC-terminal amide or ester in place of a C-terminal carboxylate; andoptionally

(4) one or more of the following modifications:

(a) covalent attachment to a hydrophilic moiety, such as polyethyleneglycol, e.g. at the N-terminus, or at position 6, 16, 17, 20, 21, 24,29, 40 or at the C-terminal amino acid; and/or

(b) acylation or alkylation; and optionally

(5) one or more of the following additional modifications:

(a) covalent linkage of amino acids, to the N-terminus, e.g. 1-5 aminoacids to the N-terminus, optionally via an ester bond to PLA at position6 (according to the numbering of wild type glucagon), optionallytogether with modifications at position 1 or 2, e.g. as describedherein, that improve resistance to DPP-IV cleavage;

(b) deletion of amino acids at positions 29 and/or 28, and optionallyposition 27 (according to the numbering of wild type glucagon);

(c) covalent linkage of amino acids to the C-terminus;

(d) non-conservative substitutions, conservative substitutions,additions or deletions while retaining desired activity, for example,conservative substitutions at one or more of positions 2, 5, 7, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29, substitution ofTyr at position 10 with Val or Phe, substitution of Lys at position 12with Arg, substitution of one or more of these positions with Ala;

(e) modification of the aspartic acid at position 15, for example, bysubstitution with glutamic acid, homoglutamic acid, cysteic acid orhomocysteic acid, which may reduce degradation; or modification of theserine at position 16, for example, by substitution of threonine, Aib,glutamic acid or with another negatively charged amino acid having aside chain with a length of 4 atoms, or alternatively with any one ofglutamine, homoglutamic acid, or homocysteic acid, which likewise mayreduce degradation due to cleavage of the Asp15-Ser16 bond;

(f) modification of the methionine at position 27, for example, bysubstitution with leucine or norleucine, to reduce oxidativedegradation;

(g) modification of the Gln at position 20 or 24, e.g. by substitutionwith Ala or Aib, to reduce degradation that occurs through deamidationof Gln

-   -   (h) modification of Asp at position 21, e.g. by substitution        with Glu, to reduce degradation that occurs through dehydration        of Asp to form a cyclic succinimide intermediate followed by        isomerization to iso-aspartate;

(j) homodimerization or heterodimerization as described herein; and

(k) combinations of the above.

It is understood that any of the modifications within the same class maybe combined together and/or modifications of different classes arecombined. For example, the modifications of (1)(a) may be combined with(2)(a) and (3); (1)(a) may be combined with (2)(b), e.g. lactam bridgeor salt bridge, and (3); (1)(a) may be combined with (2)(c) and (3);(1)(b) may be combined with (2)(a) and (3); (1)(b) may be combined with(2)(b), e.g. lactam bridge or salt bridge, and (3); (1)(b) may becombined with (2)(c) and (3); any of the foregoing may be combined with(4)(a) and/or (4)(b); and any of the foregoing may be combined with anyof (5)(a) through (5)(k).

In exemplary embodiments, the α,α-disubstituted amino acid Aib issubstituted at one, two, three or all of positions 16, 20, 21, or 24(according to the amino acid numbering of wild type glucagon).

In exemplary embodiments, the intramolecular bridge is a salt bridge.

In other exemplary embodiments, the intramolecular bridge is a covalentbond, e.g. a lactam bridge. In some embodiments, the lactam bridge isbetween the amino acids at positions 9 and 12, the amino acids atpositions 12 and 16, the amino acids at positions 16 and 20, the aminoacids at positions 20 and 24, or the amino acids at positions 24 and 28(according to the amino acid numbering of SEQ ID NO: 1401).

In exemplary embodiments, acylation or alkylation is at position 6, 10,20 or 24 or the N-terminus or C-terminus (according to the amino acidnumbering of wild type glucagon) SEQ ID NO: 1401).

In exemplary embodiments, modifications include:

(i) substitution of Asp at position 15 (according to the numbering ofSEQ ID NO: 1401) with cysteic acid, glutamic acid, homoglutamic acid,and homocysteic acid;

(ii) substitution of Ser at position 16 (according to the numbering ofSEQ ID NO: 1401) with cysteic acid, glutamic acid, homoglutamic acid,and homocysteic acid;

(iii) substitution of Asn at position 28 with a charged amino acid;

(iv) substitution of Asn at position 28 with a charged amino acidselected from the group consisting of Lys, Arg, His, Asp, Glu, cysteicacid, and homocysteic acid;

(v) substitution at position 28 with Asn, Asp, or Glu;

(vi) substitution at position 28 with Asp;

(vii) substitution at position 28 with Glu;

(viii) substitution of Thr at position 29 with a charged amino acid;

(ix) substitution of Thr at position 29 with a charged amino acidselected from the group consisting of Lys, Arg, His, Asp, Glu, cysteicacid, and homocysteic acid;

(x) substitution at position 29 with Asp, Glu, or Lys;

(xi) substitution at position 29 with Glu;

(xii) insertion of 1-3 charged amino acids after position 29;

(xiii) insertion after position 29 of Glu or Lys;

(xiv) insertion after position 29 of Gly-Lys or Lys-Lys;

or combinations thereof.

Any of the modifications described above which increase GLP-1 receptoragonist activity, glucagon receptor antagonist activity, peptidesolubility, and/or peptide stability can be applied individually or incombination.

Modification to Enhance Stability

In accordance with some embodiments the Class 5 peptides disclosedherein can be further modified to include the amino acid sequence of SEQID NO: 1421 (GPSSGAPPPS), or SEQ ID NO: 1450, linked to the carboxyterminal amino acid (position 24) of the Class 5 peptide andadministered to individuals to induce weight loss or assist in weightmaintenance. More particularly, the Class 5 peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 1405, SEQ ID NO: 1406,SEQ ID NO: 1407, SEQ ID NO: 1408, SEQ ID NO: 1409, SEQ ID NO: 1412, SEQID NO: 1413, SEQ ID NO: 1414, SEQ ID NO: 1416, SEQ ID NO: 1417, SEQ IDNO: 1418, SEQ ID NO: 1419, SEQ ID NO: 1422, SEQ ID NO: 1423, SEQ ID NO:1424 and SEQ ID NO: 1425 and further comprising the amino acid sequenceof SEQ ID NO: 1421 (GPSSGAPPPS), or SEQ ID NO: 1450, linked to thecarboxy terminal amino acid (position 24) of the peptide or Class 5peptide, is used to suppress appetite and inducing weight loss/weightmaintenance. In some embodiments the administered peptide or Class 5peptide comprises a sequence selected from the group consisting of SEQID NO: 1416, SEQ ID NO: 1417, SEQ ID NO: 1418 and SEQ ID NO: 1419,further comprising the amino acid sequence of SEQ ID NO: 1421(GPSSGAPPPS) linked to the carboxy terminal amino acid (position 24) ofthe Class 5 peptide. In some embodiments the method comprisesadministering a peptide or Class 5 peptide comprising the sequence ofSEQ ID NO: 1445 or SEQ ID NO: 1446.

Accordingly, it is expected that the Class 5 peptides disclosed hereincan be similarly modified to decrease their susceptibility to prematurechemical cleavage in aqueous buffers. In accordance with someembodiments the Class 5 peptides described herein can be furthermodified to enhance their stability in aqueous solutions by replacingthe native aspartic amino acid, located at corresponding position 15 ofnative glucagon, with an amino acid selected from the group consistingof cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid.In accordance with some embodiments the aspartic acid residue atposition 10 of class 5 peptide of SEQ ID NO: 1405, SEQ ID NO: 1406, SEQID NO: 1407 or SEQ ID NO: 1408 can be substituted with an amino acidselected from the group consisting of cysteic acid, glutamic acid,homoglutamic acid and homocysteic acid, and in some embodiments thenative aspartic acid at position 10 of SEQ ID NO: 1405, SEQ ID NO: 1406,SEQ ID NO: 1407 or SEQ ID NO: 1408 is replaced with glutamic acid. Inaccordance with some embodiments a class 5 peptide having improvedstability in aqueous solutions is provided wherein the antagonistcomprises a modified sequence of SEQ ID NO: 1409, wherein themodification comprises substitution of the Asp at position 10 of SEQ IDNO: 1409 with Glu. In some embodiments a class 5 peptide is providedcomprising a sequence selected form the group consisting of SEQ ID NO:1422, SEQ ID NO: 1423, SEQ ID NO: 1424 and SEQ ID NO: 1425. In someembodiments the class 5 peptide is amidated.

The Asp-Ser sequence at position 15-16 of native glucagon has beenidentified as a uniquely unstable dipeptide that leads to prematurechemical cleavage of the native hormone in aqueous buffers. For example,when maintained at 0.01N HCl at 37° C. for 2 weeks, more than 50% of thenative glucagon may be cleaved into fragments. The two liberatedcleavage peptides 1-15 and 16-29 are devoid of glucagon-like biologicalactivity and thus represent a limitation on the aqueous pre-formulationof glucagon and its related analogs. The selective chemical substitutionof the Asp at position 15 of native glucagon with Glu has been observedto virtually eliminate chemical cleavage of the 15-16 peptide bond.

In yet further exemplary embodiments, any of the foregoing compounds canbe further modified to improve stability by modifying the amino acidcorresponding to position 15 or 16 of native glucagon, to reducedegradation of the peptide over time, especially in acidic or alkalinebuffers.

Modification to Enhance Solubility

The class 5 peptide can be further modified to improve the peptide'ssolubility in aqueous solutions at physiological pH, in certain aspects,while retaining the glucagon antagonist and GLP-1 agonist activity.Introduction of hydrophilic groups at positions corresponding topositions 12, 15, 16, 19 and 24 of the peptide of SEQ ID NO: 1405, or atpositions 12, 16, 19 or 24 of the peptide of SEQ ID NO: 1406 can improvethe solubility of the resulting peptides in solutions having aphysiological pH, while retaining the parent compounds glucagonantagonist and GLP agonist activity. Therefore, in some embodiments thepresently disclosed class 5 peptide that are further modified tocomprise one or more hydrophilic groups covalently linked to the sidechains of amino acids corresponding to amino acid positions 12, 15, 16,19 and 24 of the peptide of SEQ ID NO: 1405 or SEQ ID NO: 1406. In afurther embodiment the side chains of amino acids corresponding to aminoacid positions 16 and 19 of SEQ ID NO: 1405 or SEQ ID NO: 1406 arecovalently bound to hydrophilic groups, and in some embodiments thehydrophilic group is polyethylene glycol (PEG).

Class 5 glucagon related peptides can be modified by introducing chargeat its carboxy terminus to enhance the solubility of the peptide whileretaining the agonist properties of the peptide. The enhanced solubilityallows for the preparation and storage of glucagon solutions at nearneutral pH. Formulating glucagon solutions at relatively neutral pHs(e.g. pH of about 6.0 to about 8.0) improves the long term stability ofthe Class 5 peptides.

Applicants anticipate that class 5 peptides disclosed herein can besimilarly modified to enhance their solubility in aqueous solutions atrelatively neutral pH (e.g. pH of about 6.0 to about 8.0), in somecases, while retaining a glucagon antagonist and GLP-1 activity.Accordingly, some embodiments is directed to a glucagon antagonist/GLP-1of SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1407 or SEQ ID NO: 1408that has been further modified relative to the native amino acidspresent at positions 6-29 of the wild type glucagon (SEQ ID NO: 1401) toadd charge to the peptide by the substitution of native non-chargedamino acids with charged amino acids, or the addition of charged aminoacids to the carboxy terminus. In accordance with some embodiments, oneto three of the non-charged native amino acids of the class 5 peptidesdisclosed herein are replaced with a charged amino acid. In someembodiments the charged amino acid is selected from the group consistingof lysine, arginine, histidine, aspartic acid and glutamic acid. Moreparticularly, applicants have discovered that substituting the normallyoccurring amino acid corresponding to position 28 and/or 29 (relative tonative glucagon) with charged amino acids, and/or the addition of one totwo charged amino acids at the carboxy terminus of the peptide, enhancesthe solubility and stability of the Class 5 peptide in aqueous solutionsat physiologically relevant pHs (i.e., a pH of about 6.5 to about 7.5).Accordingly such modifications of class 5 peptides are anticipated tohave a similar effect on the solubility in aqueous solutions,particularly at a pH ranging from about 5.5 to about 8.0, whileretaining the parent peptides biological activity.

In accordance with some embodiments the class 5 peptide of SEQ ID NO:1405, SEQ ID NO: 1406, SEQ ID NO: 1407 or SEQ ID NO: 1408 is modified bythe substitution of the native amino acid at position 23 and/or 24 ofthose sequences with a negatively charged amino acid (e.g., asparticacid or glutamic acid) and optionally the addition of a negativelycharged amino acid (e.g., aspartic acid or glutamic acid) to the carboxyterminus of the peptide. In an alternative embodiment a class 5 peptidecomprising SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1407 or SEQ IDNO: 1408 is modified by the substitution of the native amino acid atposition 24 of SEQ ID NO: 1405, SEQ ID NO: 1406, SEQ ID NO: 1407 or SEQID NO: 1408 with a positively charged amino acid (e.g., lysine, arginineor histidine) and optionally the addition of one or two positivelycharged amino acid (e.g., lysine, arginine or histidine) on the carboxyterminus of the peptide. In accordance with some embodiments a class 5peptide having improved solubility and stability is provided wherein theanalog comprises the amino acid sequence of SEQ ID NO: 1415 or SEQ IDNO: 1451 with the proviso that at least one amino acids at position, 23,or 24 of SEQ ID NO: 1415 or SEQ ID NO: 1451 is substituted with anacidic amino acid and/or an additional acidic amino acid added at thecarboxy terminus of SEQ ID NO: 1415 or SEQ ID NO: 1451. In someembodiments the acidic amino acids are independently selected from thegroup consisting of Asp, Glu, cysteic acid and homocysteic acid.

In accordance with some embodiments a class 5 peptide having improvedsolubility and stability is provided wherein the antagonist comprisesthe amino acid sequence of SEQ ID NO: 1416, SEQ ID NO: 1417, SEQ ID NO:1418 or SEQ ID NO: 1419. In accordance with some embodiments a glucagonagonist is provided comprising the sequence of SEQ ID NO: 1416 or SEQ IDNO: 1417. In some embodiments the class 5 peptide comprises the sequenceof SEQ ID NO: 1420.

In accordance with some embodiments a class 5 peptide is providedcomprising the sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451. In someembodiments, position 4 of SEQ ID NO: 1415 or SEQ ID NO: 1451 isaspartic acid, glutamic acid, homoglutamic acid, cysteic acid orhomocysteic acid, and in some embodiments position 4 is aspartic acid,glutamic acid, cysteic acid or homocysteic acid, and in a furtherembodiment position 4 of SEQ ID NO: 1415 or SEQ ID NO: 1451 is asparticacid or glutamic acid, and in some embodiments position 4 of SEQ ID NO:1415 or SEQ ID NO: 1451 is aspartic acid. In some embodiments a class 5peptide is provided comprising the sequence of SEQ ID NO: 1415 or SEQ IDNO: 1451 wherein position 4 of SEQ ID NO: 1415 is aspartic acid andposition 10 of SEQ ID NO: 1415 is glutamic acid. In a further embodimentthe C-terminal amino acid of SEQ ID NO: 1415 or SEQ ID NO: 1451 ismodified to replace the native carboxylic acid group with acharge-neutral group, such as an amide or ester.

Class 5 Peptide Fusions

In a further embodiment, the carboxy terminal amino acid of the Class 5peptide described herein is covalently bound to a second peptidecomprising a sequence selected from the group consisting of SEQ ID NOs:1421, 1426, 1427, and 1450. For example, in some embodiments, the Class5 peptide of SEQ ID NO: 1415, SEQ ID NO: 1451, SEQ ID NO: 1405, SEQ IDNO: 1406, SEQ ID NO: 1407, SEQ ID NO: 1408, SEQ ID NO: 1412, SEQ ID NO:1413, SEQ ID NO: 1414, SEQ ID NO: 1416, SEQ ID NO: 1417, SEQ ID NO:1418, SEQ ID NO: 1419, SEQ ID NO: 1422, SEQ ID NO: 1423, SEQ ID NO: 1424and SEQ ID NO: 1425 is covalently bound to a second peptide comprising asequence selected from the group consisting of SEQ ID NO: 1421(GPSSGAPPPS), SEQ ID NO: 1426 (KRNRNNIA), SEQ ID NO: 1427 (KRNR) and SEQID NO: 1450 (GPSSGAPPPSX).

In some embodiments a class 5 peptide dimer is provided comprising twosequences independently selected from the group consisting of SEQ ID NO:1405, SEQ ID NO: 1406, SEQ ID NO: 1407, SEQ ID NO: 1408, SEQ ID NO:1409, SEQ ID NO: 1422, SEQ ID NO: 1423, SEQ ID NO: 1424 and SEQ ID NO:1425 that further comprises an amino acid sequence of SEQ ID NO: 1421(GPSSGAPPPS) linked to the carboxy terminal amino acid of the class 5peptide.

In some embodiments, the class 5 peptide is further modified bytruncation or deletion of one or two amino acids of the C-terminus ofthe peptide (i.e., truncation of the amino acid at position 29 or atpositions 28 and 29 of native glucagon). Preferably truncation does noteffect activity (e.g., glucagon antagonism/GLP-1 agonism) of a class 5peptide.

Class 5 Peptide Conjugates

Conjugates of Class 5 peptides are also provided, in which the glucagonpeptide is linked, optionally via covalent bonding and optionally via alinker, to a conjugate moiety.

In those embodiments wherein the class 5 peptide comprises apolyethylene glycol chain, the polyethylene glycol chain may be in theform of a straight chain or it may be branched. In accordance with someembodiments the polyethylene glycol chain has an average molecularweight selected from the range of about 500 to about 10,000 Daltons. Insome embodiments the polyethylene glycol chain has an average molecularweight selected from the range of about 1,000 to about 5,000 Daltons. Insome embodiments the polyethylene glycol chain has an average molecularweight selected from the range of about 1,000 to about 5,000 Daltons. Insome embodiments the polyethylene glycol chain has an average molecularweight selected of about 1,000 to about 2,000 Daltons. In someembodiments the polyethylene glycol chain has an average molecularweight of about 1,000 Daltons.

In some embodiments the pegylated class 5 peptide comprises a peptideconsisting of the sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451 whereinthe polyethylene glycol chain is linked to an amino acid selected frompositions 11, 12, 15, 16, 19 and 24 of SEQ ID NO: 1415 or SEQ ID NO:1451, and the molecular weight of the PEG chain is about 1,000 to about5,000 Daltons. In some embodiments the pegylated class 5 peptidecomprises a peptide consisting of the sequence of SEQ ID NO: 1415 or SEQID NO: 1451 wherein the polyethylene glycol chain is linked to the aminoacid at position 16 or 19 of SEQ ID NO: 1415 or SEQ ID NO: 1451, and themolecular weight of the PEG chain is about 1,000 to about 5,000 Daltons.In a further embodiment the modified class 5 peptide comprises two ormore polyethylene glycol chains covalently bound to the peptide whereinthe total molecular weight of the glucagon chains is about 1,000 toabout 5,000 Daltons. In some embodiments the class 5 peptide comprisesthe sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451 wherein apolyethylene glycol chain is linked to the amino acid at positions 16and 19 of SEQ ID NO: 1415 or SEQ ID NO: 1451 and the combined molecularweight of the two PEG chains is about 1,000 to about 5,000 Daltons.

The class 5 glucagon related peptide may comprise the amino acidsequence of any of SEQ ID NOs: 1401-1518, optionally with up to 1, 2, 3,4, or 5 further modifications that retain glucagon antagonist and GLP-1agonist activity.

The Linking Group (L)

As described herein, the present disclosures provide glucagonsuperfamily peptides conjugated with GPCR ligands having the formulaQ-L-Y, wherein L is a linking group or a chemical bond. In someembodiments, L is stable in vivo. In some embodiments, L is hydrolyzablein vivo. In some embodiments, L is metastable in vivo.

Q and Y can be linked together through L using standard linking agentsand procedures known to those skilled in the art. In some aspects, Q andY are fused directly and L is a bond. In other aspects, Q and Y arefused through a linking group L. For example, in some embodiments, Q andY are linked together via a peptide bond, optionally through a peptideor amino acid spacer. In some embodiments, Q and Y are linked togetherthrough chemical conjugation, optionally through a linking group (L). Insome embodiments, L is directly conjugated to each of Q and Y.

Chemical conjugation can occur by reacting a nucleophilic reactive groupof one compound to an electrophilic reactive group of another compound.In some embodiments when L is a bond, Q is conjugated to Y either byreacting a nucleophilic reactive moiety on Q with an electrophilicreactive moiety on Y, or by reacting an electrophilic reactive moiety onQ with a nucleophilic reactive moiety on Y. In embodiments when L is agroup that links Q and Y together, Q and/or Y can be conjugated to Leither by reacting a nucleophilic reactive moiety on Q and/or Y with anelectrophilic reactive moiety on L, or by reacting an electrophilicreactive moiety on Q and/or Y with a nucleophilic reactive moiety on L.Nonlimiting examples of nucleophilic reactive groups include amino,thiol, and hydroxyl. Nonlimiting examples of electrophilic reactivegroups include carboxyl, acyl chloride, anhydride, ester, succinimideester, alkyl halide, sulfonate ester, maleimido, haloacetyl, andisocyanate. In embodiments where Q and Y are conjugated together byreacting a carboxylic acid with an amine, an activating agent can beused to form an activated ester of the carboxylic acid.

The activated ester of the carboxylic acid can be, for example,N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, acarbodiimide, or a hexafluorophosphate. In some embodiments, thecarbodiimide is 1,3-dicyclohexylcarbodiimide (DCC),1,1′-carbonyldiimidazole (CDI),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or1,3-diisopropylcarbodiimide (DICD). In some embodiments, thehexafluorophosphate is selected from a group consisting ofhexafluorophosphate benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU), ando-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU).

In some embodiments, Q comprises a nucleophilic reactive group (e.g. theamino group, thiol group, or hydroxyl group of the side chain of lysine,cysteine or serine) that is capable of conjugating to an electrophilicreactive group on Y or L. In some embodiments, Q comprises anelectrophilic reactive group (e.g. the carboxylate group of the sidechain of Asp or Glu) that is capable of conjugating to a nucleophilicreactive group on Y or L. In some embodiments, Q is chemically modifiedto comprise a reactive group that is capable of conjugating directly toY or to L. In some embodiments, Q is modified at the C-terminal tocomprise a natural or nonnatural amino acid with a nucleophilic sidechain, such as an amino acid represented by Formula I, Formula II, orFormula III, as previously described herein (see Acylation andalkylation). In exemplary embodiments, the C-terminal amino acid of Q isselected from the group consisting of lysine, ornithine, serine,cysteine, and homocysteine. For example, the C-terminal amino acid of Qcan be modified to comprise a lysine residue. In some embodiments, Q ismodified at the C-terminal amino acid to comprise a natural ornonnatural amino acid with an electrophilic side chain such as, forexample, Asp and Glu. In some embodiments, an internal amino acid of Qis substituted with a natural or nonnatural amino acid having anucleophilic side chain, such as an amino acid represented by Formula I,Formula II, or Formula III, as previously described herein (seeAcylation and alkylation). In exemplary embodiments, the internal aminoacid of Q that is substituted is selected from the group consisting oflysine, ornithine, serine, cysteine, and homocysteine. For example, aninternal amino acid of Q can be substituted with a lysine residue. Insome embodiments, an internal amino acid of Q is substituted with anatural or nonnatural amino acid with an electrophilic side chain, suchas, for example, Asp and Glu.

In some embodiments, Y comprises a reactive group that is capable ofconjugating directly to Q or to L. In some embodiments, Y comprises anucleophilic reactive group (e.g. amine, thiol, hydroxyl) that iscapable of conjugating to an electrophilic reactive group on Q or L. Insome embodiments, Y comprises electrophilic reactive group (e.g.carboxyl group, activated form of a carboxyl group, compound with aleaving group) that is capable of conjugating to a nucleophilic reactivegroup on Q or L. In some embodiments, Y is chemically modified tocomprise either a nucleophilic reactive group that is capable ofconjugating to an electrophilic reactive group on Q or L. In someembodiments, Y is chemically modified to comprise an electrophilicreactive group that is capable of conjugating to a nucleophilic reactivegroup on Q or L.

In some embodiments, conjugation can be carried out throughorganosilanes, e.g., aminosilane treated with glutaraldehyde;carbonyldiimidazole (CDI) activation of silanol groups; or utilizationof dendrimers. A variety of dendrimers are known in the art and includepoly (amidoamine) (PAMAM) dendrimers, which are synthesized by thedivergent method starting from ammonia or ethylenediamine initiator corereagents; a sub-class of PAMAM dendrimers based on atris-aminoethylene-imine core; radially layeredpoly(amidoamine-organosilicon) dendrimers (PAMAMOS), which are invertedunimolecular micelles that consist of hydrophilic, nucleophilicpolyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS)exteriors; Poly (Propylene Imine) (PPI) dendrimers, which are generallypoly-alkyl amines having primary amines as end groups, while thedendrimer interior consists of numerous of tertiary tris-propyleneamines; Poly (Propylene Amine) (POPAM) dendrimers; Diaminobutane (DAB)dendrimers; amphiphilic dendrimers; micellar dendrimers which areunimolecular micelles of water soluble hyper branched polyphenylenes;polylysine dendrimers; and dendrimers based on poly-benzyl ether hyperbranched skeleton.

In some embodiments, conjugation can be carried out through olefinmetathesis. In some embodiments, Y and Q, Y and L, or Q and L bothcomprise an alkene or alkyne moiety that is capable of undergoingmetathesis. In some embodiments a suitable catalyst (e.g. copper,ruthenium) is used to accelerate the metathesis reaction. Suitablemethods of performing olefin metathesis reactions are described in theart. See, for example, Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000), Walensky et al., Science 305: 1466-1470 (2004), andBlackwell et al., Angew, Chem., Int. Ed. 37: 3281-3284 (1998).

In some embodiments, conjugation can be carried out using clickchemistry. A “click reaction” is wide in scope and easy to perform, usesonly readily available reagents, and is insensitive to oxygen and water.In some embodiments, the click reaction is a cycloaddition reactionbetween an alkynyl group and an azido group to form a triazolyl group.In some embodiments, the click reaction uses a copper or rutheniumcatalyst. Suitable methods of performing click reactions are describedin the art. See, for example, Kolb et al., Drug Discovery Today 8:1128(2003); Kolb et al., Angew. Chem. Int. Ed. 40:2004 (2001); Rostovtsev etal., Angew. Chem. Int. Ed. 41:2596 (2002); Tornoe et al., J. Org. Chem.67:3057 (2002); Manetsch et al., J. Am. Chem. Soc. 126:12809 (2004);Lewis et al., Angew. Chem. Int. Ed. 41:1053 (2002); Speers, J. Am. Chem.Soc. 125:4686 (2003); Chan et al. Org. Lett. 6:2853 (2004); Zhang etal., J. Am. Chem. Soc. 127:15998 (2005); and Waser et al., J. Am. Chem.Soc. 127:8294 (2005).

Indirect conjugation via high affinity specific binding partners, e.g.streptavidin/biotin or avidin/biotin or lectin/carbohydrate is alsocontemplated.

Chemical Modification of Q and/or Y

In some embodiments, Q and/or Y are functionalized to comprise anucleophilic reactive group or an electrophilic reactive group with anorganic derivatizing agent. This derivatizing agent is capable ofreacting with selected side chains or the N- or C-terminal residues oftargeted amino acids on Q and functional groups on Y. Reactive groups onQ and/or Y include, e.g., aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group. Derivatizing agents include, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride or other agents known in the art.Alternatively, Q and/or Y can be linked to each other indirectly throughintermediate carriers, such as polysaccharide or polypeptide carriers.Examples of polysaccharide carriers include aminodextran. Examples ofsuitable polypeptide carriers include polylysine, polyglutamic acid,polyaspartic acid, co-polymers thereof, and mixed polymers of theseamino acids and others, e.g., serines, to confer desirable solubilityproperties on the resultant loaded carrier.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin.

Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagine or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the peptide. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Structure of L

In some embodiments, L is a bond. In these embodiments, Q and Y areconjugated together by reacting a nucleophilic reactive moiety on Q withand electrophilic reactive moiety on Y. In alternative embodiments, Qand Y are conjugated together by reacting an electrophilic reactivemoiety on Q with a nucleophilic moiety on Y. In exemplary embodiments, Lis an amide bond that forms upon reaction of an amine on Q (e.g. anε-amine of a lysine residue) with a carboxyl group on Y. In alternativeembodiments, Q and or Y are derivatized with a derivatizing agent beforeconjugation.

In some embodiments, L is a linking group. In some embodiments, L is abifunctional linker and comprises only two reactive groups beforeconjugation to Q and Y. In embodiments where both Q and Y haveelectrophilic reactive groups, L comprises two of the same or twodifferent nucleophilic groups (e.g. amine, hydroxyl, thiol) beforeconjugation to Q and Y. In embodiments where both Q and Y havenucleophilic reactive groups, L comprises two of the same or twodifferent electrophilic groups (e.g. carboxyl group, activated form of acarboxyl group, compound with a leaving group) before conjugation to Qand Y. In embodiments where one of Q or Y has a nucleophilic reactivegroup and the other of Q or Y has an electrophilic reactive group, Lcomprises one nucleophilic reactive group and one electrophilic groupbefore conjugation to Q and Y.

L can be any molecule with at least two reactive groups (beforeconjugation to Q and Y) capable of reacting with each of Q and Y. Insome embodiments L has only two reactive groups and is bifunctional. L(before conjugation to the peptides) can be represented by Formula VI:

wherein A and B are independently nucleophilic or electrophilic reactivegroups. In some embodiments A and B are either both nucleophilic groupsor both electrophilic groups. In some embodiments one of A or B is anucleophilic group and the other of A or B is an electrophilic group.Nonlimiting combinations of A and B are shown below.

Both Nucleophilic Both Electrophilic Nucleophilic/Electrophilic A B A BA B amino Amino carboxyl carboxyl amino carboxyl amino Thiol carboxylacyl chloride amino acyl chloride amino hydroxyl carboxyl anhydrideamino anhydride thiol Amino carboxyl Ester amino ester thiol Thiolcarboxyl NHS amino NHS thiol hydroxyl carboxyl Halogen amino halogenhydroxyl Amino carboxyl sulfonate ester amino sulfonate ester hydroxylThiol carboxyl maleimido amino maleimido hydroxyl hydroxyl carboxylhaloacetyl amino haloacetyl carboxyl isocyanate amino isocyanate acylchloride carboxyl thiol carboxyl acyl chloride acyl chloride thiol acylchloride acyl chloride anhydride thiol anhydride acyl chloride Esterthiol ester acyl chloride NHS thiol NHS acyl chloride Halogen thiolhalogen acyl chloride sulfonate ester thiol sulfonate ester acylchloride maleimido thiol maleimido acyl chloride haloacetyl thiolhaloacetyl acyl chloride isocyanate thiol isocyanate anhydride carboxylhydroxyl carboxyl anhydride acyl chloride hydroxyl acyl chlorideanhydride anhydride hydroxyl anhydride anhydride Ester hydroxyl esteranhydride NHS hydroxyl NHS anhydride Halogen hydroxyl halogen anhydridesulfonate ester hydroxyl sulfonate ester anhydride maleimido hydroxylmaleimido anhydride haloacetyl hydroxyl haloacetyl anhydride isocyanatehydroxyl isocyanate ester carboxyl ester acyl chloride ester anhydrideester Ester ester NHS ester Halogen ester sulfonate ester estermaleimido ester haloacetyl ester isocyanate NHS carboxyl NHS acylchloride NHS anhydride NHS Ester NHS NHS NHS Halogen NHS sulfonate esterNHS maleimido NHS haloacetyl NHS isocyanate halogen carboxyl halogenacyl chloride halogen anhydride halogen Ester halogen NHS halogenHalogen halogen sulfonate ester halogen maleimido halogen haloacetylhalogen isocyanate sulfonate ester carboxyl sulfonate ester acylchloride sulfonate ester anhydride sulfonate ester Ester sulfonate esterNHS sulfonate ester Halogen sulfonate ester sulfonate ester sulfonateester maleimido sulfonate ester haloacetyl sulfonate ester isocyanatemaleimido carboxyl maleimido acyl chloride maleimido anhydride maleimidoEster maleimido NHS maleimido Halogen maleimido sulfonate estermaleimido maleimido maleimido haloacetyl maleimido isocyanate haloacetylcarboxyl haloacetyl acyl chloride haloacetyl anhydride haloacetyl Esterhaloacetyl NHS haloacetyl Halogen haloacetyl sulfonate ester haloacetylmaleimido haloacetyl haloacetyl haloacetyl isocyanate isocyanatecarboxyl isocyanate acyl chloride isocyanate anhydride isocyanate Esterisocyanate NHS isocyanate Halogen isocyanate sulfonate ester isocyanatemaleimido isocyanate haloacetyl isocyanate isocyanateIn some embodiments, A and B may include alkene and/or alkyne functionalgroups that are suitable for olefin metathesis reactions. In someembodiments, A and B include moieties that are suitable for clickchemistry (e.g. alkene, alkynes, nitriles, azides). Other nonlimitingexamples of reactive groups (A and B) include pyridyldithiol, arylazide, diazirine, carbodiimide, and hydrazide.

In some embodiments, L is hydrophobic. Hydrophobic linkers are known inthe art. See, e.g., Bioconjugate Techniques, G. T. Hermanson (AcademicPress, San Diego, Calif., 1996), which is incorporated by reference inits entirety. Suitable hydrophobic linking groups known in the artinclude, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid. Before conjugation to the peptides of the composition, thehydrophobic linking group comprises at least two reactive groups (A andB), as described herein and as shown below:

In some embodiments, the hydrophobic linking group comprises either amaleimido or an iodoacetyl group and either a carboxylic acid or anactivated carboxylic acid (e.g. NHS ester) as the reactive groups. Inthese embodiments, the maleimido or iodoacetyl group can be coupled to athiol moiety on Q or Y and the carboxylic acid or activated carboxylicacid can be coupled to an amine on Q or Y with or without the use of acoupling reagent. Any coupling agent known to one skilled in the art canbe used to couple the carboxylic acid with the free amine such as, forexample, DCC, DIC, HATU, HBTU, TBTU, and other activating agentsdescribed herein. In specific embodiments, the hydrophilic linking groupcomprises an aliphatic chain of 2 to 100 methylene groups wherein A andB are carboxyl groups or derivatives thereof (e.g. succinic acid). Inother specific embodiments the L is iodoacetic acid.

In some embodiments, the linking group is hydrophilic such as, forexample, polyalkylene glycol. Before conjugation to the peptides of thecomposition, the hydrophilic linking group comprises at least tworeactive groups (A and B), as described herein and as shown below:

In specific embodiments, the linking group is polyethylene glycol (PEG).The PEG in certain embodiments has a molecular weight of about 100Daltons to about 10,000 Daltons, e.g. about 500 Daltons to about 5000Daltons. The PEG in some embodiments has a molecular weight of about10,000 Daltons to about 40,000 Daltons.

In some embodiments, the hydrophilic linking group comprises either amaleimido or an iodoacetyl group and either a carboxylic acid or anactivated carboxylic acid (e.g. NHS ester) as the reactive groups. Inthese embodiments, the maleimido or iodoacetyl group can be coupled to athiol moiety on Q or Y and the carboxylic acid or activated carboxylicacid can be coupled to an amine on Q or Y with or without the use of acoupling reagent. Any appropriate coupling agent known to one skilled inthe art can be used to couple the carboxylic acid with the amine suchas, for example, DCC, DIC, HATU, HBTU, TBTU, and other activating agentsdescribed herein In some embodiments, the linking group ismaleimido-PEG(20 kDa)-COOH, iodoacetyl-PEG(20 kDa)-COOH,maleimido-PEG(20 kDa)-NHS, or iodoacetyl-PEG(20 kDa)-NHS.

In some embodiments, the linking group is comprised of an amino acid, adipeptide, a tripeptide, or a polypeptide, wherein the amino acid,dipeptide, tripeptide, or polypeptide comprises at least two activatinggroups, as described herein. In some embodiments, the linking group (L)comprises a moiety selected from the group consisting of: amino, ether,thioether, maleimido, disulfide, amide, ester, thioester, alkene,cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsinB-cleavable, and hydrazone.

In some embodiments, L comprises a chain of atoms from 1 to about 60, or1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or10 to 20 atoms long. In some embodiments, the chain atoms are all carbonatoms. In some embodiments, the chain atoms in the backbone of thelinker are selected from the group consisting of C, O, N, and S. Chainatoms and linkers may be selected according to their expected solubility(hydrophilicity) so as to provide a more soluble conjugate. In someembodiments, L provides a functional group that is subject to cleavageby an enzyme or other catalyst or hydrolytic conditions found in thetarget tissue or organ or cell. In some embodiments, the length of L islong enough to reduce the potential for steric hindrance.

Stability of L In Vivo

In some embodiments, L is stable in vivo. In some embodiments, L isstable in blood serum for at least 5 minutes, e.g. less than 25%, 20%,15%, 10% or 5% of the conjugate is cleaved when incubated in serum for aperiod of 5 minutes. In other embodiments, L is stable in blood serumfor at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 24 hours. In these embodiments, Ldoes not comprise a functional group that is capable of undergoinghydrolysis in vivo. In some exemplary embodiments, L is stable in bloodserum for at least about 72 hours.

In some embodiments, L is hydrolyzable in vivo. In these embodiments, Lcomprises a functional group that is capable of undergoing hydrolysis invivo. Nonlimiting examples of functional groups that are capable ofundergoing hydrolysis in vivo include esters, anhydrides, andthioesters. For example the following compound is capable of undergoinghydrolysis in vivo because it comprises an ester group:

In some exemplary embodiments L is labile and undergoes substantialhydrolysis within 3 hours in blood plasma at 37° C., with completehydrolysis within 6 hours. In some exemplary embodiments, L is notlabile.

Nonlimiting examples of functional groups that are not capable ofundergoing significant hydrolysis in vivo include amides, ethers, andthioethers. For example, the following compound is not capable ofundergoing significant hydrolysis in vivo:

In some embodiments, L is metastable in vivo. In these embodiments, Lcomprises a functional group that is capable of being chemically orenzymatically cleaved in vivo (e.g., an acid-labile, reduction-labile,or enzyme-labile functional group), optionally over a period of time. Inthese embodiments, L can comprise, for example, a hydrazone moiety, adisulfide moiety, or a cathepsin-cleavable moiety. When L is metastable,and without intending to be bound by any particular theory, the Q-L-Yconjugate is stable in an extracellular environment, e.g., stable inblood serum for the time periods described above, but labile in theintracellular environment or conditions that mimic the intracellularenvironment, so that it cleaves upon entry into a cell. In someembodiments when L is metastable, L is stable in blood serum for atleast about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or48 hours, for example, at least about 48, 54, 60, 66, or 72 hours, orabout 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

Q-L-Y Conjugates

Conjugation of Q and Y

Conjugation of Q to Y through L can be carried out an any positionwithin Q, including any of positions 1-29, a position within aC-terminal extension, or the C-terminal amino acid, provided that theactivity of Q is retained, if not enhanced. Nonlimiting examples includepositions 5, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27,28, 29, 30, 37, 38, 39, 40, 41, 42, or 43 (according to the number ofthe amino acids of SEQ ID NO: 1601). In some embodiments, Y isconjugated to Q through L at one or more of positions 10, 20, 24, 30,37, 38, 39, 40, 41, 32, or 43. In specific embodiments, Y is conjugatedto Q through L at position 10 and/or 40 of Q.

Activity

Activity at the Glucagon Receptor and the GPCR

In some embodiments, Q-L-Y exhibits activity at both the glucagonreceptor and a GPCR. In some embodiments, the activity (e.g., the EC₅₀or the relative activity or potency) of Q at the glucagon receptor iswithin about 100-fold, about 75-fold, about 60-fold, about 50-fold,about 40-fold, about 30-fold, about 20-fold, about 10-fold, or about 5fold different (higher or lower) from the activity (e.g., the EC₅₀ orthe relative activity or potency) of Y at a GPCR. In some embodiments,the glucagon potency of Q is within about 25-, about 20-, about 15-,about 10-, or about 5-fold different (higher or lower) from the potencyof Y.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the glucagon receptor divided by the relativeactivity or the EC₅₀ or potency of Y at a GPCR is less than, or isabout, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the glucagon receptor divided by the EC₅₀ orpotency or relative activity of Y at a GPCR is about 1 less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, theratio of the glucagon potency of Q compared to the GPCR activatingpotency of Y is less than, or is about, Z, wherein Z is selected from100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In some embodiments, theratio of the glucagon potency of Q compared to the GPCR activatingpotency of Y is less than 5 (e.g., about 4, about 3, about 2, about 1).In some embodiments, Q has an EC₅₀ at the glucagon receptor which is 2-to 10-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold) greater than the EC₅₀ of Y at a GPCR.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Y at a GPCR divided by the relative activity or potency orthe EC₅₀ of Q at the glucagon receptor is less than, or is about, V,wherein V is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. Insome embodiments, the ratio of the EC₅₀ or potency or relative activityof Y at a GPCR divided by the EC₅₀ or potency or relative activity of Qat the glucagon receptor is less than 5 (e.g., about 4, about 3, about2, about 1). In some embodiments, the ratio of the GPCR activatingpotency of Y compared to the glucagon potency of Q is less than, or isabout, W, wherein W is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, and 5. In some embodiments, the ratio of the GPCR activating potencyof Y compared to the glucagon potency of Q is less than 5 (e.g., about4, about 3, about 2, about 1). In some embodiments, Y has an EC₅₀ at aGPCR which is about 2- to about 10-fold (e.g., 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greater than the EC₅₀of Q at the glucagon receptor.

In some embodiments, Y exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of an endogenous ligand at a GPCR (GPCR activating potency) andQ exhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeglucagon at the glucagon receptor (glucagon potency).

Activity at the GLP-1 Receptor and the GPCR

In some embodiments, Q-L-Y exhibits activity at both the GLP-1 receptorand a GPCR. In some embodiments, the activity (e.g., the EC₅₀ or therelative activity or potency) of Q at the GLP-1 receptor is within about100-fold, about 75-fold, about 60-fold, about 50-fold, about 40-fold,about 30-fold, about 20-fold, about 10-fold, or about 5 fold different(higher or lower) from the activity (e.g., the EC₅₀ or the relativeactivity or potency) of Y at a GPCR. In some embodiments, the GLP-1potency of Q is within about 25-, about 20-, about 15-, about 10-, orabout 5-fold different (higher or lower) from the potency of Y.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the GLP-1 receptor divided by the relativeactivity or the EC₅₀ or potency of Y at a GPCR is less than, or isabout, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the GLP-1 receptor divided by the EC₅₀ orpotency or relative activity of Y at a GPCR is about 1 less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, theratio of the GLP-1 potency of Q compared to the GPCR activating potencyof Y is less than, or is about, Z, wherein Z is selected from 100, 75,60, 50, 40, 30, 20, 15, 10, and 5. In some embodiments, the ratio of theGLP-1 potency of Q compared to the GPCR activating potency of Y is lessthan 5 (e.g., about 4, about 3, about 2, about 1). In some embodiments,Q has an EC₅₀ at the GLP-1 receptor which is 2- to 10-fold (e.g.,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold)greater than the EC₅₀ of Y at a GPCR.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Y at a GPCR divided by the relative activity or potency orthe EC₅₀ of Q at the GLP-1 receptor is less than, or is about, V,wherein V is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. Insome embodiments, the ratio of the EC₅₀ or potency or relative activityof Y at a GPCR divided by the EC₅₀ or potency or relative activity of Qat the GLP-1 receptor is less than 5 (e.g., about 4, about 3, about 2,about 1). In some embodiments, the ratio of the GPCR activating potencyof Y compared to the GLP-1 potency of Q is less than, or is about, W,wherein W is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, and 5.In some embodiments, the ratio of the GPCR activating potency of Ycompared to the GLP-1 potency of Q is less than 5 (e.g., about 4, about3, about 2, about 1). In some embodiments, Y has an EC₅₀ at a GPCR whichis about 2- to about 10-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greater than the EC₅₀ of Q atthe GLP-1 receptor.

In some embodiments, Y exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of endogenous ligand at a GPCR (GPCR activating potency) and Qexhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeGLP-1 at the GLP-1 receptor (GLP-1 potency).

Activity at the GIP Receptor and the GPCR

In some embodiments, Q-L-Y exhibits activity at both the GIP receptorand a GPCR. In some embodiments, the activity (e.g., the EC₅₀ or therelative activity or potency) of Q at the GIP receptor is within about100-fold, about 75-fold, about 60-fold, about 50-fold, about 40-fold,about 30-fold, about 20-fold, about 10-fold, or about 5 fold different(higher or lower) from the activity (e.g., the EC₅₀ or the relativeactivity or potency) of Y at a GPCR. In some embodiments, the GIPpotency of Q is within about 25-, about 20-, about 15-, about 10-, orabout 5-fold different (higher or lower) from the potency of Y.

In some embodiments, the ratio of the relative activity or the EC₅₀ orthe potency of the Q at the GIP receptor divided by the relativeactivity or the EC₅₀ or potency of Y at a GPCR is less than, or isabout, X, wherein X is selected from 100, 75, 60, 50, 40, 30, 20, 15,10, or 5. In some embodiments, the ratio of the EC₅₀ or potency orrelative activity of Q at the GIP receptor divided by the EC₅₀ orpotency or relative activity of Y at a GPCR is about 1 less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, theratio of the GIP potency of Q compared to the GPCR activating potency ofY is less than, or is about, Z, wherein Z is selected from 100, 75, 60,50, 40, 30, 20, 15, 10, and 5. In some embodiments, the ratio of the GIPpotency of Q compared to the GPCR activating potency of Y is less than 5(e.g., about 4, about 3, about 2, about 1). In some embodiments, Q hasan EC₅₀ at the GIP receptor which is 2- to 10-fold (e.g., 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold) greaterthan the EC₅₀ of Y at a GPCR.

In some embodiments, the ratio of the relative activity or potency orthe EC₅₀ of Y at a GPCR divided by the relative activity or potency orthe EC₅₀ of Q at the GIP receptor is less than, or is about, V, whereinV is selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In someembodiments, the ratio of the EC₅₀ or potency or relative activity of Yat a GPCR divided by the EC₅₀ or potency or relative activity of Q atthe GIP receptor is less than 5 (e.g., about 4, about 3, about 2, about1). In some embodiments, the ratio of the GPCR activating potency of Ycompared to the GIP potency of Q is less than, or is about, W, wherein Wis selected from 100, 75, 60, 50, 40, 30, 20, 15, 10, and 5. In someembodiments, the ratio of the GPCR activating potency of Y compared tothe GIP potency of Q is less than 5 (e.g., about 4, about 3, about 2,about 1). In some embodiments, Y has an EC₅₀ at a GPCR which is about 2-to about 10-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold) greater than the EC₅₀ of Q at the GIP receptor.

In some embodiments, Y exhibits at least 0.1% (e.g., about 0.5% or more,about 1% or more, about 5% or more, about 10% or more, or more) of theactivity of endogenous ligand at a GPCR (GPCR activating potency) and Qexhibits at least 0.1% (e.g., about 0.5% or more, about 1% or more,about 5% or more, about 10% or more, or more) of the activity of nativeGIP at the GIP receptor (GIP potency).

Prodrugs of Q-L-Y

In some aspects of the invention, prodrugs of Q-L-Y are provided whereinthe prodrug comprises a dipeptide prodrug element (A-B) covalentlylinked to an active site of Q via an amide linkage, as disclosed inInternational Patent Application No. PCT US09/68745 (filed on Dec. 18,2009), which is incorporated herein by reference in its entirety.Subsequent removal of the dipeptide under physiological conditions andin the absence of enzymatic activity, restores full activity to theQ-L-Y conjugate.

In some embodiments a prodrug of Q-L-Y is provided having the generalstructure of A-B-Q-L-Y. In these embodiments A is an amino acid or ahydroxy acid and B is an N-alkylated amino acid linked to Q throughformation of an amide bond between a carboxyl of B (in A-B) and an amineof Q. Furthermore, in some embodiments, A, B, or the amino acid of Q towhich A-B is linked, is a non-coded amino acid, and chemical cleavage ofA-B from Q is at least about 90% complete within about 1 to about 720hours in PBS under physiological conditions. In another embodiment,chemical cleavage of A-B from Q is at least about 50% complete withinabout 1 hour or about 1 week in PBS under physiological conditions.

In some embodiment the dipeptide prodrug element (A-B) comprises acompound having the general structure below:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In some embodiments, the dipeptide prodrug element is linked to theamino terminus of Q. In other embodiments, the dipeptide prodrug islinked to an internal amino acid of Q, as described in InternationalPatent Application No. PCT US09/68745.

The conjugates comprising the glucagon superfamily peptides of theinvention (Q) and a GPCR ligand (Y) have synergistic pharmacology. The Qportion of these conjugates can act to direct the GPCR portion of theconjugate to targets of desired action to result in improved glycemiccontrol (e.g. as measured by decreased glucose levels) and energyhomeostasis (e.g. as measured by decreased body weight and/or fat mass),with an enhanced therapeutic index.

For example, in diet-induced obese mice, a fully active GLP-1 agonistwith a stably-linked GPCR are more efficacious in reducing blood glucoseand body weight than the comparative GLP-1 control, and the combinedpresence of GLP-1 and targeted GPCR can achieve superior reductions inglucose and body weight.

The “meta”-stable peptide-GPCR conjugates are stable in plasma butcapable of releasing a GPCR ligand upon cellular internalization. Theseglucagon superfamily peptide/GPCR conjugates with metastable linkagesare able to lower glucose levels and body weight to a greater extentthan conjugates with labile linkages.

In exemplary embodiments, the conjugates are useful for treating any ofthe conditions described herein, including but not limited to ahyperglycemic medical condition, obesity, metabolic syndrome, and NAFLD.

Pharmaceutical Compositions

Salts

In some embodiments, the Q-L-Y conjugates described herein are in theform of a salt, e.g., a pharmaceutically acceptable salt. As used hereinthe term “pharmaceutically acceptable salt” refers to salts of compoundsthat retain the biological activity of the parent compound, and whichare not biologically or otherwise undesirable. Such salts can beprepared in situ during the final isolation and purification of theconjugate, or separately prepared by reacting a free base function witha suitable acid. Many of the compounds disclosed herein are capable offorming acid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Representative acid addition salts include,but are not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate,hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived frominorganic acids include hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. Salts derived fromorganic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Examples of acidswhich can be employed to form pharmaceutically acceptable acid additionsalts include, for example, an inorganic acid, e.g., hydrochloric acid,hydrobromic acid, sulphuric acid, and phosphoric acid, and an organicacid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the source of salicylic acid, or byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate, or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia or an organic primary,secondary, or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on alkali metals or alkaline earthmetals such as lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like, and nontoxic quaternary ammonia and aminecations including ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like. Salts derived from organic bases include, butare not limited to, salts of primary, secondary and tertiary amines.

Further, basic nitrogen-containing groups can be quaternized with theconjugate of the present disclosure as lower alkyl halides such asmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; longchain halides such as decyl, lauryl, myristyl, and stearyl chlorides,bromides, and iodides; arylalkyl halides like benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

Formulations

In accordance with some embodiments, a pharmaceutical composition isprovided wherein the composition comprises a Q-L-Y conjugate of thepresent disclosure, or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The pharmaceutical composition cancomprise any pharmaceutically acceptable ingredient, including, forexample, acidifying agents, additives, adsorbents, aerosol propellants,air displacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers, filmforming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one ora combination of the following components: acacia, acesulfame potassium,acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol,dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid,alginic acid, aliphatic polyesters, alumina, aluminum hydroxide,aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbylpalmitate, aspartame, bacteriostatic water for injection, bentonite,bentonite magma, benzalkonium chloride, benzethonium chloride, benzoicacid, benzyl alcohol, benzyl benzoate, bronopol, butylatedhydroxyanisole, butylated hydroxytoluene, butylparaben, butylparabensodium, calcium alginate, calcium ascorbate, calcium carbonate, calciumcyclamate, dibasic anhydrous calcium phosphate, dibasic dehydratecalcium phosphate, tribasic calcium phosphate, calcium propionate,calcium silicate, calcium sorbate, calcium stearate, calcium sulfate,calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium,β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC)chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capable of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g., at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Routes of Administration

The following discussion on routes of administration is merely providedto illustrate exemplary embodiments and should not be construed aslimiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the conjugate of the presentdisclosure dissolved in diluents, such as water, saline, or orangejuice; (b) capsules, sachets, tablets, lozenges, and troches, eachcontaining a predetermined amount of the active ingredient, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations may include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant. Capsule forms can be of theordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers, such as lactose, sucrose,calcium phosphate, and corn starch. Tablet forms can include one or moreof lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the conjugate of the presentdisclosure in a flavor, usually sucrose and acacia or tragacanth, aswell as pastilles comprising the conjugate of the present disclosure inan inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to, suchexcipients as are known in the art.

The conjugates of the disclosure, alone or in combination with othersuitable components, can be delivered via pulmonary administration andcan be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa. In some embodiments, theconjugate is formulated into a powder blend or into microparticles ornanoparticles. Suitable pulmonary formulations are known in the art.See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei andGarren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al.,J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res10(2): 228-232 (1993); International Patent Application Publication Nos.WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The conjugate of the present disclosure can be administeredwith a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol or hexadecyl alcohol, a glycol, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of Q-L-Y conjugate of the present disclosure insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene glycol sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the conjugate of the present disclosures can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the conjugate of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dose

The Q-L-Y conjugates of the disclosure are believed to be useful inmethods of treating a disease or medical condition in which glucagonreceptor agonism, GLP-1 receptor agonism, GIP receptor agonism, glucagonreceptor/GLP-1 receptor co-agonism, glucagon receptor/GIP receptorco-agonism, GLP-1 receptor/GIP receptor co-agonism or glucagonreceptor/GLP-1 receptor/GIP receptor tri-agonism plays a role. Forpurposes of the disclosure, the amount or dose of the conjugate of thepresent disclosure administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the conjugate of thepresent disclosure should be sufficient to stimulate cAMP secretion fromcells as described herein or sufficient to decrease blood glucoselevels, fat levels, food intake levels, or body weight of a mammal, in aperiod of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks orlonger, e.g., 5 to 20 or more weeks, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular conjugate of the presentdisclosure and the condition of the animal (e.g., human), as well as thebody weight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich blood glucose levels are lowered upon administration of a givendose of the conjugate of the present disclosure to a mammal among a setof mammals of which is each given a different dose of the conjugate,could be used to determine a starting dose to be administered to amammal. The extent to which blood glucose levels are lowered uponadministration of a certain dose can be assayed by methods known in theart.

The dose of the conjugate of the present disclosure also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particularconjugate of the present disclosure. Typically, the attending physicianwill decide the dosage of the conjugate of the present disclosure withwhich to treat each individual patient, taking into consideration avariety of factors, such as age, body weight, general health, diet, sex,conjugate of the present disclosure to be administered, route ofadministration, and the severity of the condition being treated. By wayof example and not intending to limit the invention, the dose of theconjugate of the present disclosure can be about 0.0001 to about 1 g/kgbody weight of the subject being treated/day, from about 0.0001 to about0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day.

In some embodiments, the pharmaceutical composition comprises any of theconjugates disclosed herein at a purity level suitable foradministration to a patient. In some embodiments, the conjugate has apurity level of at least about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, anda pharmaceutically acceptable diluent, carrier or excipient. Thepharmaceutical composition in some aspects comprise the conjugate of thepresent disclosure at a concentration of at least A, wherein A is about0.001 mg/ml, about 0.01 mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml,about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml,about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24mg/ml, about 25 mg/ml or higher. In some embodiments, the pharmaceuticalcomposition comprises the conjugate at a concentration of at most B,wherein B is about 30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23,mg/ml, about 22 mg/ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml,about 18 mg/ml, about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14mg/ml, about 13 mg/ml, about 12 mg/ml, about 11 mg/ml, about 10 mg/ml,about 9 mg/ml, about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5mg/ml, about 4 mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, orabout 0.1 mg/ml. In some embodiments, the compositions may contain anconjugate at a concentration range of A to B mg/ml, for example, about0.001 to about 30.0 mg/ml.

Targeted Forms

One of ordinary skill in the art will readily appreciate that the Q-L-Yconjugates of the disclosure can be modified in any number of ways, suchthat the therapeutic or prophylactic efficacy of the conjugate of thepresent disclosures is increased through the modification. For instance,the conjugate of the present disclosure can be further conjugated eitherdirectly or indirectly through a linker to a targeting moiety. Thepractice of conjugating compounds, e.g., glucagon conjugates describedherein, to targeting moieties is known in the art. See, for instance,Wadhwa et al., J Drug Targeting, 3, 111-127 (1995) and U.S. Pat. No.5,087,616. The term “targeting moiety” as used herein, refers to anymolecule or agent that specifically recognizes and binds to acell-surface receptor, such that the targeting moiety directs thedelivery of the conjugate of the present disclosures to a population ofcells on which surface the receptor (the glucagon receptor, the GLP-1receptor) is expressed. Targeting moieties include, but are not limitedto, antibodies, or fragments thereof, peptides, hormones, growthfactors, cytokines, and any other natural or non-natural ligands, whichbind to cell surface receptors (e.g., Epithelial Growth Factor Receptor(EGFR), T-cell receptor (TCR), B-cell receptor (BCR), CD28,Platelet-derived Growth Factor Receptor (PDGF), nicotinic acetylcholinereceptor (nAChR), etc.). As used herein a “linker” is a bond, moleculeor group of molecules that binds two separate entities to one another.Linkers may provide for optimal spacing of the two entities or mayfurther supply a labile linkage that allows the two entities to beseparated from each other. Labile linkages include photocleavablegroups, acid-labile moieties, base-labile moieties, hydrolyzable groups,and enzyme-cleavable groups. The term “linker” in some embodimentsrefers to any agent or molecule that bridges the conjugate of thepresent disclosures to the targeting moiety. One of ordinary skill inthe art recognizes that sites on the conjugate of the presentdisclosures, which are not necessary for the function of the conjugateof the present disclosures, are ideal sites for attaching a linkerand/or a targeting moiety, provided that the linker and/or targetingmoiety, once attached to the conjugate of the present disclosures,do(es) not interfere with the function of the conjugate of the presentdisclosures, i.e., the ability to stimulate cAMP secretion from cells,to treat diabetes or obesity. One of ordinary skill in the artrecognizes that sites on the peptide of the present disclosures (Q),which are not necessary for the function of the peptide of the presentdisclosures (e.g., glucagon agonist peptide, glucagon antagonistpeptide, GLP-1 agonist peptide, GIP agonist peptide, or a combination ofany of the foregoing), are ideal sites for attaching a linker and/or atargeting moiety, provided that the linker and/or targeting moiety, onceattached to the peptide of the present disclosures (Q), does notinterfere with the function of the peptide of the present disclosures.

Controlled Release Formulations

Alternatively, the glucagon conjugates described herein can be modifiedinto a depot form, such that the manner in which the conjugate of thepresent disclosures is released into the body to which it isadministered is controlled with respect to time and location within thebody (see, for example, U.S. Pat. No. 4,450,150). Depot forms ofconjugate of the present disclosures can be, for example, an implantablecomposition comprising the conjugate of the present disclosures and aporous or non-porous material, such as a polymer, wherein the conjugateof the present disclosures is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body and theconjugate of the present disclosures are released from the implant at apredetermined rate.

The pharmaceutical composition in certain aspects is modified to haveany type of in vivo release profile. In some aspects, the pharmaceuticalcomposition is an immediate release, controlled release, sustainedrelease, extended release, delayed release, or bi-phasic releaseformulation. Methods of formulating peptides or conjugates forcontrolled release are known in the art. See, for example, Qian et al.,J Pharm 374: 46-52 (2009) and International Patent ApplicationPublication Nos. WO 2008/130158, WO2004/033036; WO2000/032218; and WO1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. The disclosed pharmaceutical formulations may be administeredaccording to any regime including, for example, daily (1 time per day, 2times per day, 3 times per day, 4 times per day, 5 times per day, 6times per day), every two days, every three days, every four days, everyfive days, every six days, weekly, bi-weekly, every three weeks,monthly, or bi-monthly.

Uses

Based on the information provided for the first time herein, it iscontemplated that the Q-L-Y conjugates described herein and relatedpharmaceutical compositions are useful for treatment of a disease ormedical condition, in which e.g., the lack of activity at the glucagonreceptor, the GLP-1 receptor, the GIP receptor or a combination of anyof the foregoing, is a factor in the onset and/or progression of thedisease or medical condition. Accordingly, the invention provides amethod of treating or preventing a disease or medical condition in apatient, wherein the disease or medical condition is a disease ofmedical condition in which a lack of GLP-1 receptor activation and/orglucagon receptor activation and/or GIP activation is associated withthe onset and/or progression of the disease of medical condition. Themethod comprises providing to the patient an conjugate in accordancewith any of those described herein in an amount effective to treat orprevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolicsyndrome. Metabolic Syndrome, also known as metabolic syndrome X,insulin resistance syndrome or Reaven's syndrome, is a disorder thataffects over 50 million Americans. Metabolic Syndrome is typicallycharacterized by a clustering of at least three or more of the followingrisk factors: (1) abdominal obesity (excessive fat tissue in and aroundthe abdomen), (2) atherogenic dyslipidemia (blood fat disordersincluding high triglycerides, low HDL cholesterol and high LDLcholesterol that enhance the accumulation of plaque in the arterywalls), (3) elevated blood pressure, (4) insulin resistance or glucoseintolerance, (5) prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in blood), and (6) pro-inflammatorystate (e.g., elevated C-reactive protein in blood). Other risk factorsmay include aging, hormonal imbalance and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronaryheart disease and other disorders related to the accumulation ofvascular plaque, such as stroke and peripheral vascular disease,referred to as atherosclerotic cardiovascular disease (ASCVD). Patientswith Metabolic Syndrome may progress from an insulin resistant state inits early stages to full blown type II diabetes with further increasingrisk of ASCVD. Without intending to be bound by any particular theory,the relationship between insulin resistance, Metabolic Syndrome andvascular disease may involve one or more concurrent pathogenicmechanisms including impaired insulin-stimulated vasodilation, insulinresistance-associated reduction in NO availability due to enhancedoxidative stress, and abnormalities in adipocyte-derived hormones suchas adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B-76B(2004)).

According to the 2001 National Cholesterol Education Program AdultTreatment Panel (ATP III), any three of the following traits in the sameindividual meet the criteria for Metabolic Syndrome: (a) abdominalobesity (a waist circumference over 102 cm in men and over 88 cm inwomen); (b) serum triglycerides (150 mg/dl or above); (c) HDLcholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women);(d) blood pressure (130/85 or more); and (e) fasting blood glucose (110mg/dl or above). According to the World Health Organization (WHO), anindividual having high insulin levels (an elevated fasting blood glucoseor an elevated post meal glucose alone) with at least two of thefollowing criteria meets the criteria for Metabolic Syndrome: (a)abdominal obesity (waist to hip ratio of greater than 0.9, a body massindex of at least 30 kg/m2, or a waist measurement over 37 inches); (b)cholesterol panel showing a triglyceride level of at least 150 mg/dl oran HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 ormore, or on treatment for high blood pressure). (Mathur, Ruchi,“Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May11, 2009).

For purposes herein, if an individual meets the criteria of either orboth of the criteria set forth by the 2001 National CholesterolEducation Program Adult Treatment Panel or the WHO, that individual isconsidered as afflicted with Metabolic Syndrome.

Without being bound to any particular theory, Q-L-Y conjugates describedherein are useful for treating Metabolic Syndrome. Accordingly, theinvention provides a method of preventing or treating MetabolicSyndrome, or reducing one, two, three or more risk factors thereof, in asubject, comprising providing to the subject a conjugate describedherein in an amount effective to prevent or treat Metabolic Syndrome, orthe risk factor thereof.

In some embodiments, the method treats a hyperglycemic medicalcondition. In certain aspects, the hyperglycemic medical condition isdiabetes, diabetes mellitus type I, diabetes mellitus type II, orgestational diabetes, either insulin-dependent or non-insulin-dependent.In some aspects, the method treats the hyperglycemic medical conditionby reducing one or more complications of diabetes including nephropathy,retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In someaspects, the obesity is drug-induced obesity. In some aspects, themethod treats obesity by preventing or reducing weight gain orincreasing weight loss in the patient. In some aspects, the methodtreats obesity by reducing appetite, decreasing food intake, loweringthe levels of fat in the patient, or decreasing the rate of movement offood through the gastrointestinal system.

Because obesity is associated with the onset or progression of otherdiseases, the methods of treating obesity are further useful in methodsof reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases. The invention accordinglyprovides methods of treating or preventing these obesity-associatedcomplications.

In some embodiments, the disease or medical condition is Nonalcoholicfatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liverdisease ranging from simple fatty liver (steatosis), to nonalcoholicsteatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring ofthe liver). All of the stages of NAFLD have in common the accumulationof fat (fatty infiltration) in the liver cells (hepatocytes). Simplefatty liver is the abnormal accumulation of a certain type of fat,triglyceride, in the liver cells with no inflammation or scarring. InNASH, the fat accumulation is associated with varying degrees ofinflammation (hepatitis) and scarring (fibrosis) of the liver. Theinflammatory cells can destroy the liver cells (hepatocellularnecrosis). In the terms “steatohepatitis” and “steatonecrosis”, steatorefers to fatty infiltration, hepatitis refers to inflammation in theliver, and necrosis refers to destroyed liver cells. NASH can ultimatelylead to scarring of the liver (fibrosis) and then irreversible, advancedscarring (cirrhosis). Cirrhosis that is caused by NASH is the last andmost severe stage in the NAFLD spectrum. (Mendler, Michel, “Fatty Liver:Nonalcoholic Fatty Liver Disease (NAFLD) and NonalcoholicSteatohepatitis (NASH),” ed. Schoenfield, Leslie J., MedicineNet.com,Aug. 29, 2005).

Alcoholic Liver Disease, or Alcohol-Induced Liver Disease, encompassesthree pathologically distinct liver diseases related to or caused by theexcessive consumption of alcohol: fatty liver (steatosis), chronic oracute hepatitis, and cirrhosis. Alcoholic hepatitis can range from amild hepatitis, with abnormal laboratory tests being the only indicationof disease, to severe liver dysfunction with complications such asjaundice (yellow skin caused by bilirubin retention), hepaticencephalopathy (neurological dysfunction caused by liver failure),ascites (fluid accumulation in the abdomen), bleeding esophageal varices(varicose veins in the esophagus), abnormal blood clotting and coma.Histologically, alcoholic hepatitis has a characteristic appearance withballooning degeneration of hepatocytes, inflammation with neutrophilsand sometimes Mallory bodies (abnormal aggregations of cellularintermediate filament proteins). Cirrhosis is characterized anatomicallyby widespread nodules in the liver combined with fibrosis. (Worman,Howard J., “Alcoholic Liver Disease”, Columbia University Medical Centerwebsite).

Without being bound to any particular theory, the Q-L-Y conjugatesdescribed herein are useful for the treatment of Alcoholic LiverDisease, NAFLD, or any stage thereof, including, for example, steatosis,steatohepatitis, hepatitis, hepatic inflammation, NASH, cirrhosis, orcomplications thereof. Accordingly, the invention provides a method ofpreventing or treating Alcoholic Liver Disease, NAFLD, or any stagethereof, in a subject comprising providing to a subject a conjugatedescribed herein in an amount effective to prevent or treat AlcoholicLiver Disease, NAFLD, or the stage thereof. Such treatment methodsinclude reduction in one, two, three or more of the following: liver fatcontent, incidence or progression of cirrhosis, incidence ofhepatocellular carcinoma, signs of inflammation, e.g., abnormal hepaticenzyme levels (e.g., aspartate aminotransferase AST and/or alanineaminotransferase ALT, or LDH), elevated serum ferritin, elevated serumbilirubin, and/or signs of fibrosis, e.g., elevated TGF-beta levels. Inpreferred embodiments, the Q-L-Y conjugates described herein are usedtreat patients who have progressed beyond simple fatty liver (steatosis)and exhibit signs of inflammation or hepatitis. Such methods may result,for example, in reduction of AST and/or ALT levels.

GLP-1 and exendin-4 have been shown to have some neuroprotective effect.The invention also provides uses of the Q-L-Y conjugates describedherein in treating neurodegenerative diseases, including but not limitedto Alzheimer's disease, Parkinson's disease, Multiple Sclerosis,Amylotrophic Lateral Sclerosis, other demyelination related disorders,senile dementia, subcortical dementia, arteriosclerotic dementia,AIDS-associated dementia, or other dementias, a central nervous systemcancer, traumatic brain injury, spinal cord injury, stroke or cerebralischemia, cerebral vasculitis, epilepsy, Huntington's disease,Tourette's syndrome, Guillain Barre syndrome, Wilson disease, Pick'sdisease, neuroinflammatory disorders, encephalitis, encephalomyelitis ormeningitis of viral, fungal or bacterial origin, or other centralnervous system infections, prion diseases, cerebellar ataxias,cerebellar degeneration, spinocerebellar degeneration syndromes,Friedreichs ataxia, ataxia telangiectasia, spinal dysmyotrophy,progressive supranuclear palsy, dystonia, muscle spasticity, tremor,retinitis pigmentosa, striatonigral degeneration, mitochondrialencephalo-myopathies, neuronal ceroid lipofuscinosis, hepaticencephalopathies, renal encephalopathies, metabolic encephalopathies,toxin-induced encephalopathies, and radiation-induced brain damage.

In some embodiments, the disease or medical condition is hypoglycemia.In some embodiments, the patient is a diabetic patient and thehypoglycemia is induced by the administration of insulin. In specificaspects, the method comprises providing the conjugate of the presentdisclosure in combination with insulin so that the conjugate buffers thehypoglycemic effects of the bolus administration of insulin.

In some embodiments, the Q-L-Y conjugates are used in conjunction withparenteral administration of nutrients to non-diabetic patients in ahospital setting, e.g., to patients receiving parenteral nutrition ortotal parenteral nutrition. Nonlimiting examples include surgerypatients, patients in comas, patients with digestive tract illness, or anonfunctional gastrointestinal tract (e.g. due to surgical removal,blockage or impaired absorptive capacity, Crohn's disease, ulcerativecolitis, gastrointestinal tract obstruction, gastrointestinal tractfistula, acute pancreatitis, ischemic bowel, major gastrointestinalsurgery, certain congenital gastrointestinal tract anomalies, prolongeddiarrhea, or short bowel syndrome due to surgery, patients in shock, andpatients undergoing healing processes often receive parenteraladministration of carbohydrates along with various combinations oflipids, electrolytes, minerals, vitamins and amino acids. The Q-L-Yconjugates and the parenteral nutrition composition can be administeredat the same time, at different times, before, or after each other,provided that the Q-L-Y conjugate is exerting the desired biologicaleffect at the time that the parenteral nutrition composition is beingdigested. For example, the parenteral nutrition may be administered, 1,2 or 3 times per day, while the glucagon conjugate is administered onceevery other day, three times a week, two times a week, once a week, onceevery 2 weeks, once every 3 weeks, or once a month.

As used herein, the terms “treat,” and “prevent” as well as wordsstemming therefrom, do not necessarily imply 100% or complete treatmentor prevention. Rather, there are varying degrees of treatment orprevention of which one of ordinary skill hi the art recognizes ashaving a potential benefit or therapeutic effect. In this respect, theinventive methods can provide any amount of any level of treatment orprevention of a disease or medical condition in a mammal. Furthermore,the treatment or prevention provided by the method can include treatmentor prevention of one or more conditions or symptoms of the disease ormedical condition. For example, with regard to methods of treatingobesity, the method in some embodiments, achieves a decrease in foodintake by or fat levels in a patient. Also, for purposes herein,“prevention” can encompass delaying the onset of the disease, or asymptom or condition thereof.

With regard to the above methods of treatment, the patient is any host.In some embodiments, the host is a mammal. As used herein, the term“mammal” refers to any vertebrate animal of the mammalia class,including, but not limited to, any of the monotreme, marsupial, andplacental taxas. In some embodiments, the mammal is one of the mammalsof the order Rodentia, such as mice and hamsters, and mammals of theorder Logomorpha, such as rabbits. In certain embodiments, the mammalsare from the order Carnivora, including Felines (cats) and Canines(dogs). In certain embodiments, the mammals are from the orderArtiodactyla, including Bovines (cows) and S wines (pigs) or of theorder Perssodactyla, including Equines (horses). In some instances, themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). In particular embodiments, themammal is a human.

Combinations

The Q-L-Y conjugates described herein may be administered alone or incombination with other therapeutic agents which aim to treat or preventany of the diseases or medical conditions described herein. For example,the Q-L-Y conjugates described herein may be co-administered with(simultaneously or sequentially) an anti-diabetic or anti-obesity agent.Anti-diabetic agents known in the art or under investigation includeinsulin, leptin, Peptide YY (PYY), Pancreatic Peptide (PP), fibroblastgrowth factor 21 (FGF21), Y2Y4 receptor agonists, sulfonylureas, such astolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta,Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron);meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix);biguanides such as metformin (Glucophage) or phenformin;thiazolidinediones such as rosiglitazone (Avandia), pioglitazone(Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alphaglucosidase inhibitors that inhibit carbohydrate digestion, such asmiglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) orpramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such asvildagliptin or sitagliptin; SGLT (sodium-dependent glucosetransporter 1) inhibitors; glucokinase activators (GKA); glucagonreceptor antagonists (GRA); or FBPase (fructose 1,6-bisphosphatase)inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®), Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to XENICAL (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

The Q-L-Y conjugates described herein in some embodiments areco-administered with an agent for treatment of non-alcoholic fatty liverdisease or NASH. Agents used to treat non-alcoholic fatty liver diseaseinclude ursodeoxycholic acid (a.k.a., Actigall, URSO, and Ursodiol),Metformin (Glucophage), rosiglitazone (Avandia), Clofibrate,Gemfibrozil, Polymixin B, and Betaine.

The Q-L-Y conjugates described herein in some embodiments areco-administered with an agent for treatment of a neurodegenerativedisease, e.g., Parkinson's Disease. Anti-Parkinson's Disease agents arefurthermore known in the art and include, but not limited to, levodopa,carbidopa, anticholinergics, bromocriptine, pramipexole, and ropinirole,amantadine, and rasagiline.

In view of the foregoing, the invention further provides pharmaceuticalcompositions and kits additionally comprising one of these othertherapeutic agents. The additional therapeutic agent may be administeredsimultaneously or sequentially with the conjugate of the presentdisclosure. In some aspects, the conjugate is administered before theadditional therapeutic agent, while in other aspects, the conjugate isadministered after the additional therapeutic agent.

Kits

The Q-L-Y conjugates of the present disclosure can be provided inaccordance with one embodiment as part of a kit. Accordingly, in someembodiments, a kit for administering a Q-L-Y conjugate to a patient inneed thereof is provided wherein the kit comprises a Q-L-Y conjugate asdescribed herein.

In one embodiment the kit is provided with a device for administeringthe Q-L-Y conjugate composition to a patient, e.g. syringe needle, pendevice, jet injector or other needle-free injector. The kit mayalternatively or in addition include one or more containers, e.g.,vials, tubes, bottles, single or multi-chambered pre-filled syringes,cartridges, infusion pumps (external or implantable), jet injectors,pre-filled pen devices and the like, optionally containing the glucagonconjugate in a lyophilized form or in an aqueous solution. The kits insome embodiments comprise instructions for use. In accordance with oneembodiment the device of the kit is an aerosol dispensing device,wherein the composition is prepackaged within the aerosol device. Inanother embodiment the kit comprises a syringe and a needle, and in oneembodiment the sterile glucagon composition is prepackaged within thesyringe.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1 Synthesis of Peptide Fragments of GlucagonSuperfamily Peptides

Materials

Peptides are synthesized by any procedures known in the art. Exemplaryprotocols follow. Peptides can be amidated at the C-terminus. MBHA(4-methylbenzhydrylamine polystyrene) resin can be used during peptidesynthesis. MBHA resin, 100-180 mesh, 1% divinylbenzene (DVB)cross-linked polystyrene; loading of 0.7-1.0 mmol/g), Boc-protected(tert-butylcarbamates) and Fmoc protected (9-fluoenylmethyl carbamate)amino acids can be purchased from Midwest Biotech. The solid phasepeptide syntheses using Boc-protected amino acids can be performed on anApplied Biosystem 430A Peptide Synthesizer. Fmoc protected amino acidsynthesis can be performed using the Applied Biosystems Model 433Peptide Synthesizer. In specific embodiments, the following procedurescan be used:

General Peptide Synthesis Protocol with Boc-Chemistry Strategy

Synthesis of peptides using Boc chemistry can be performed on theApplied Biosystem Model 430A Peptide Synthesizer. Synthetic peptides areconstructed by sequential addition of amino acids to a cartridgecontaining 2 mmol of Boc protected amino acid. Specifically, thesynthesis can be carried out using3-(Diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one (DEPBT) orHBTU as the coupling agent with single couplings. At the end of thecoupling step, the peptidyl-resin is treated with trifluoroacetic acid(TFA) to remove the N-terminal Boc protecting group. The resin is thenwashed repeatedly with dimethylformamide (DMF) and this repetitive cycleis repeated for the desired number of coupling steps. Boc amino acidsand HBTU can be obtained from Midwest Biotech (Fishers, Ind.). Generalside chain protecting groups that can be used include: Arg(Tos),Asn(Xan), Asp(OcHex), Cys(pMeBzl), His(Bom), Lys(2Cl-Z), Ser(OBzl),Thr(OBzl), Tyr(2Br-Z), and Trp(CHO). Boc-Glu(OFm)-OH andBoc-Lys(Fmoc)-OH (Chem-Impex, Wood dale, IL) is used in thelactam-bridge formation sites.

After assembly of the peptides, the Fmoc protected side chains aredeprotected using 20% piperidine treatment. For the lactamization,orthogonal protecting groups are selected for Glu and Lys (e.g.,Glu(Fm), Lys(Fmoc)). After removal of the protecting groups and beforeHF cleavage, cyclization is performed as described previously (see,e.g., International Patent Application Publication No. WO2008/101017).HF treatment of the peptidyl-resin

The peptidyl-resin is treated with anhydrous HF in the presence ofp-cresol and dimethyl sulfide, which typically yielded approximately 350mg (about 50% yield) of a crude deprotected-peptide. Specifically, thepeptidyl-resin (30 mg to 200 mg) is placed in a hydrogen fluoride (HF)reaction vessel for cleavage. Then, 500 μL of p-cresol is added to thevessel as a carbonium ion scavenger. The vessel is attached to the HFsystem and submerged in a methanol/dry ice mixture. The vessel isevacuated with a vacuum pump and 10 mL of HF was distilled into thereaction vessel. This reaction mixture of the peptidyl-resin and the HFwas stirred for one hour at 0° C., after which a vacuum was establishedand the HF is quickly evacuated (10-15 min) The vessel is then removedcarefully and filled with approximately 35 mL of ether to precipitatethe peptide and to extract the p-cresol and small molecule organicprotecting groups resulting from HF treatment. This mixture is filteredutilizing a Teflon filter and repeated twice to remove all excesscresol. This filtrate is discarded. The precipitated peptide isdissolved in approximately 20 mL of 10% acetic acid (aq). This filtrate,which contained the desired peptide, is collected and lyophilized.

An analytical HPLC analysis of the crude solubilized peptide isconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% TFA/10% acetonitrile (CH₃CN), B buffer0.1% TFA/100% CH₃CN, gradient 5-95% B over 15 minutes]. The extract isdiluted twofold with water and loaded onto a 2.2×25 cm Vydac C4preparative reverse phase column and eluted using an acetonitrilegradient on a Waters HPLC system (A buffer of 0.1% TFA/10% CH₃CN, Bbuffer of 0.1% TFA/10% CH₃CN and a gradient of 0-100% B over 120 minutesat a flow of 15.00 mL/min. HPLC analysis of the purified peptidedemonstrated greater than 95% purity and electrospray ionization massspectral analysis can be used to confirm the identity of the peptide.

General Peptide Synthesis Protocol with Fmoc-Chemistry Strategy:

Peptides can be synthesized on an ABI 433A automated peptide synthesizerusing standard Fmoc chemistry with Rink MBHA amide resin or first aminoacid attached Wang resin (Novabiochem, San Diego, Calif.) using DIC/HOBTas coupling reagent. The side chain protecting groups of Na-Fmoc[N-(9-fluorenyl)methoxycarbonyl]amino acids that can be used include thefollowing: Arg, Pmc; Asp, OtBu; Cys, Trt; Gln, Trt; His, Trt; Lys, Boc;Ser, tBu, Tyr, tBu; and Trp, Boc(Pmc=2,2,5,7,8-pentamethylchoman-6-sulfonyl, OtBu32 tert-butyl ester,Trt=trityl, Boc32 tert-butyloxycarbonyl, and tBu32 tert-butyl ester).Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)-OH (Novabiochem, San Diego,Calif.) are incorporated in the lactam-bridge formation sites.

After solid phase synthesis, the 2-phenylisopropyl (2-PhiPr) group onthe Glu and the 4-methoxytrityl (Mmt) group on the Lys are removed byflashing 1% TFA/DCM though the peptidyl resin. For the lactam-bridgeformation, usually 150 mg (0.5 mmole, 5-fold) BEPBT are added in 10%DIEA/DMF and reacted for 2 to 4 h until ninhydrin test shown negative.

Peptides are cleaved from the resin with cleavage cocktail containing85% TFA, 5% phenol, 5% water and 5% thioanisole (2.5% EDT is added whenpeptide contains Cysteine). Crude peptides are precipitated in ether,centrifuged, and lyophilized. Peptides are then analyzed by analyticalHPLC and checked by ESI or MALDI-TOF mass spectrometry. Peptides arethen purified by the general HPLC purification procedure.

Peptide Acylation

Acylated peptides can be prepared as follows. Peptides are synthesizedon a solid support resin using either a CS Bio 4886 Peptide Synthesizeror Applied Biosystems 430A Peptide Synthesizer. In situ neutralizationchemistry can be used as described by Schnolzer et al., Int. J. PeptideProtein Res. 40: 180-193 (1992). For acylated peptides, the target aminoacid residue to be acylated (e.g., position ten, relative to the aminoacid position numbering of SEQ ID NO: 1601) is substituted with anNε-FMOC lysine residue. Treatment of the completed N-terminally BOCprotected peptide with 20% piperidine in DMF for 30 minutes is used toremove FMOC/formyl groups. Coupling to the free ε-amino Lys residue isachieved by reacting a ten-fold molar excess of either an FMOC-protectedspacer amino acid (ex. FMOC-Glu-OtBu) or acyl chain (e.g.CH₃(CH₂)₁₄—COOH) and benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP), N,N′diisopropylcarbodiimide (DIC), or DEPBTcoupling reagent in DMF/diisopropylethylamine (DIEA). Subsequent removalof the spacer amino acid's FMOC group is followed by repetition ofcoupling with an acyl chain. Final treatment with 100% TFA resulted inremoval of any side chain protecting groups and the N-terminal BOCgroup. Peptide resins are neutralized with 5% DIEA/DMF, dried, and thencleaved from the support using HF/p-cresol, 95:5, at 0° C. for one hour.Following ether extraction, a 5% acetic acid (HOAc) solution is used tosolvate the crude peptide. A sample of the solution was then verified tocontain the correct molecular weight peptide by ESI-MS. Correct peptidesare typically purified by reverse phase (RP) HPLC using a lineargradient of 10% CH₃CN/0.1% TFA to 0.1% TFA in 100% CH₃CN. A Vydac C18 22mm×250 mm protein column can be used for the purification. Acylatedpeptide conjugates generally completed elution by a buffer ratio of20:80. Portions can be pooled together and checked for purity on ananalytical RP-HPLC. Pure fractions are lyophilized yielding white, solidpeptides.

If a peptide comprised a lactam bridge and target residues to beacylated, acylation can be carried out as described above upon additionof that amino acid to the peptide backbone.

Peptide PEGylation to Form Thioether Linkages

For peptide PEGylation, 40 kDa methoxy poly(ethylene glycol)idoacetamide is reacted with a molar equivalent of peptide in 7M Urea,50 mM Tris-HCl buffer using the minimal amount of solvent needed todissolve both peptide and PEG into a clear solution (generally less than2 mL for a reaction using 2 to 3 mg peptide). Vigorous stirring at roomtemperature is commenced for 4 to 6 hours and the reaction can beanalyzed by analytical RP-HPLC. PEGylated products appeared distinctlyfrom the starting material with decreased retention times. Purificationcan be performed on a Vydac C4 column with conditions similar to thoseused for the initial peptide purification. Elution occurrs around bufferratios of 50:50. Fractions of pure PEGylated peptide are isolated andlyophilized. Yields will be above 50%, varying per reaction.

Peptide PEGylation to Form Maleimido Linkages

For peptide PEGylation, the peptide containing a cysteine is dissolvedin phosphate buffered saline (5- to 0 mg/mL) and 0.01 M ethylenediaminetetraacetic acid is added (10-15% of total volume). Excess (2-fold)maleimido methoxyPEG reagent (Dow) is added and the reaction is stirredat room temperature while monitoring reaction progress by highperformance liquid chromatography (HPLC). After 8-24 hrs, the reactionmixture is acidified and loaded onto a preparative reverse phase columnfor purification using 0.1% tetrafluoroacetic acid (TFA)/acetonitrile inthe gradient mode. The appropriate fractions are combined andlyophilized to give the desired pegylated derivatives.

Analysis Using Mass Spectrometry

Mass spectra are obtained using a Sciex API-III electrospray quadrapolemass spectrometer with a standard ESI ion source. Ionization conditionsthat are used are as follows: ESI in the positive-ion mode; ion sprayvoltage, 3.9 kV; orifice potential, 60 V. The nebulizing and curtain gasused is nitrogen flow rate of 0.9 L/min. Mass spectra are recorded from600-1800 Thompsons at 0.5 Th per step and 2 msec dwell time. The sample(about lmg/mL) is dissolved in 50% aqueous acetonitrile with 1% aceticacid and introduced by an external syringe pump at the rate of 5 μL/min

When the peptides are analyzed in PBS solution by ESI MS, they are firstdesalted using a ZipTip solid phase extraction tip containing 0.6 μL C4resin, according to instructions provided by the manufacturer (MilliporeCorporation, Billerica, Mass., see the Millipore website of the worldwide web at millipore.com/catalogue.nsf/docs/C5737).

High Performance Liquid Chromatography (HPLC) Analysis:

Preliminary analyses are performed with these crude peptides to get anapproximation of their relative conversion rates in Phosphate BufferedSaline (PBS) buffer (pH, 7.2) using high performance liquidchromatography (HPLC) and MALDI analysis. The crude peptide samples aredissolved in the PBS buffer at a concentration of 1 mg/mL. 1 mL of theresulting solution is stored in a 1.5 mL HPLC vial which is then sealedand incubated at 37° C. Aliquots of 100 μl are drawn out at various timeintervals, cooled to room temperature and analyzed by HPLC.

The HPLC analyses are performed using a Beckman System GoldChromatography system using a UV detector at 214 nm HPLC analyses areperformed on a 150 mm×4.6 mm C18 Vydac column. The flow rate is 1mL/min. Solvent A contained 0.1% TFA in distilled water, and solvent Bcontained 0.1% TFA in 90% CH₃CN. A linear gradient is employed (40% to70% B in 15 minutes). The data are collected and analyzed using PeakSimple Chromatography software.

Example 2

The ability of each peptide to induce cAMP can be measured in a fireflyluciferase-based reporter assay. The cAMP production that is induced isdirectly proportional to the glucagon fragment binding to the glucagonreceptor or GIP receptor or GLP-1 receptor. HEK293 cells co-transfectedwith the receptor and luciferase gene linked to a cAMP responsiveelement are employed for the bioassay.

The cells are serum-deprived by culturing 16 hours in Dulbecco-modifiedMinimum Essential Medium (Invitrogen, Carlsbad, Calif.) supplementedwith 0.25% Bovine Growth Serum (HyClone, Logan, Utah) and then incubatedwith serial dilutions of glucagon fragments for 5 hours at 37° C., 5%CO₂ in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation, 100 μL of LucLiteluminescence substrate reagent (Perkin Elmer, Wellesley, Mass.) is addedto each well. The plate is shaken briefly, incubated 10 min in the darkand light output was measured on MicroBeta-1450 liquid scintillationcounter (Perkin-Elmer, Wellesley, Mass.). The effective 50%concentrations (EC₅₀) and inhibitory 50% concentrations (IC₅₀) arecalculated by using Origin software (OriginLab, Northampton, Mass.).

Example 3

Diet-induced obesity mice are administered subcutaneous injections onceper day for two weeks with vehicle or 40 or 400 μg/kg of one of thefollowing:

(a) GLP-1 agonist,

(b) GLP-1 agonist stably linked to Y,

(c) GLP-1 agonist with a labile linkage to Y, or

(d) GLP-1 agonist with a metastable linkage to Y.

Body weight is measured after 14 days and the change in body weight isdetermined. Mice that are administered the GLP-1 agonist conjugatesexperience a greater decrease in body weight than mice that areadministered the GLP-1 agonist alone.

The effect of the conjugates on cumulative food intake over a period of14 days is also determined Mice that are administered the GLP-1 agonistconjugates consume less food than mice that are administered the GLP-1agonist alone.

The effect of the conjugates on the change in blood glucose isdetermined. Mice that are administered the GLP-1 agonist conjugatesexperience a greater decrease in blood glucose levels over the course ofthe study than mice that are administered the GLP-1 agonist alone.

Example 4

Diet-induced obesity mice are administered subcutaneous injections onceper day for two weeks with vehicle or 40 or 400 μg/kg of one of thefollowing:

(a) GLP-1 agonist,

(b) GLP-1 agonist stably linked to a β3 adrenergic receptor agonist,

(c) GLP-1 agonist with a labile linkage to a β3 adrenergic receptoragonist,

(d) GLP-1 agonist stably linked to a nicotinic acetylcholine receptoragonist, or (e) GLP-1 agonist with a labile linkage to a nicotinicacetylcholine receptor agonist.

Body weight is measured after 14 days and the change in body weight isdetermined. Mice that are administered the GLP-1 agonist conjugatesexperience a greater decrease in body weight than mice that areadministered the GLP-1 agonist alone.

The effect of the conjugates on cumulative food intake over a period of14 days is also determined Mice that are administered the GLP-1 agonistconjugates consume less food than mice that are administered the GLP-1agonist alone.

The effect of the conjugates on the change in blood glucose isdetermined Mice that are administered the GLP-1 agonist conjugatesexperience a greater decrease in blood glucose levels over the course ofthe study than mice that are administered the GLP-1 agonist alone.

MEGA

The invention claimed is:
 1. A compound comprising the structure Q-L-Y;wherein Q is a glucagon superfamily peptide having agonist activity atthe glucagon receptor, the GIP receptor, the GLP-1 receptor, or acombination thereof; Y is a G protein-coupled receptor (GPCR) ligandwhich activates a G protein-coupled receptor selected from an adrenergicreceptor or a nicotinic acetylcholine receptor with an EC₅₀ of about 1μM or less, and has a molecular weight of up to about 1000 daltons; andL is a linking group or a bond, wherein Q comprises an amino acidsequence at least 50% identical to native glucagon that retains thealpha-helix conformation of the amino acids corresponding to amino acids12-29 of native glucagon (SEQ ID NO: 1601) or an amino acid sequence atleast 50% identical to native GLP-1 that retains the alpha-helixconformation of the amino acids corresponding to amino acids 12-29 ofnative GLP-1 (SEQ ID NO: 1603); wherein L-Y is conjugated to Q atposition 10, 20, 24, 30, 37, 38, 39, 40, 41, 32, or 43 of Q.
 2. Thecompound of claim 1, wherein Q exhibits at least 0.1% of the activity ofnative glucagon-like peptide-1 (GLP-1) at the GLP-1 receptor, Q exhibitsat least 0.1% of the activity of native glucagon at the glucagonreceptor, or Q exhibits at least 0.1% of the activity of native gastricinhibitory polypeptide (GIP) at the GIP receptor, or a combinationthereof.
 3. The compound of claim 1, wherein Q has an EC₅₀ at the GLP-1receptor within 10-fold of the EC₅₀ of Y at the G protein-coupledreceptor, Q has an EC₅₀ at the glucagon receptor within 10-fold of theEC₅₀ of Y at the G protein-coupled receptor, or Q has an EC₅₀ at the GIPreceptor within 10-fold of the EC₅₀ of Y at the G protein-coupledreceptor, or a combination thereof.
 4. The compound of claim 1, whereinL is stable in vivo, hydrolyzable in vivo, or metastable in vivo.
 5. Thecompound of claim 4, wherein L comprises an ether moiety, an amidemoiety, an ester moiety, an acid-labile moiety, a reduction-labilemoiety, an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety,or a cathepsin-cleavable moiety.
 6. The compound of claim 1, wherein Qcomprises (I) the amino acid sequence:X₁-X₂-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z(SEQ ID NO: 839) with 1 to 3 amino acid modifications thereto, whereinX_(i) and/or X₂ is a non-native amino acid (relative to SEQ ID NO: 1601)that reduces susceptibility of Q to cleavage by dipeptidyl peptidase IV(DPP-IV), wherein Z is selected from the group consisting of —COOH,-Asn-COOH, Asn-Thr-COOH, and W—COOH, wherein W is 1 to 2 amino acids orGPSSGAPPPS (SEQ ID NO: 1610), wherein Q comprises a modificationselected from the group consisting of: (i) a lactam bridge between theside chains of amino acids at positions i and i+4, wherein i is 12, 16,20 or 24, and (ii) one, two, three, or all of the amino acids atpositions 16, 20, 21, and 24 is substituted with an α,α-disubstitutedamino acid; and, wherein Q exhibits glucagon agonist activity; or, (II)the amino acid sequence of SEQ ID NO: 1601 and comprises: (a) at leastone amino acid modification selected from the group consisting of: (i)substitution of Thr at position 29 with a charged amino acid; (ii)substitution of Thr at position 29 with a charged amino acid selectedfrom the group consisting of Lys, Arg, His, Asp, Glu, cysteic acid, andhomocysteic acid; (iii) substitution at position 29 with Asp, Glu, orLys; (iv) substitution at position 29 with Glu; (v) insertion afterposition 29 of 1 to 3 charged amino acids; (vi) insertion after position29 of Glu or Lys; (vii) insertion after position 29 of Gly-Lys, orLys-Lys; (viii) substitution of Gln at position 3 with an amino acidcomprising a side chain of Structure I, II, or III:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃ alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, O, or S; and Y isNHR⁴, SR³, or OR³; and (ix) a combination thereof; and, (b) substitutionof Ser at position 16 with Thr, Glu, or Aib; and at least one amino acidmodification selected from the group consisting of: (i) substitution ofHis at position 1 with a non-native amino acid that reducessusceptibility of the Q to cleavage by dipeptidyl peptidase IV (DPP-IV),(ii) substitution of Ser at position 2 with a non-native amino acid thatreduces susceptibility of the Q to cleavage by dipeptidyl peptidase IV(DPP-IV), (iii) substitution of Thr at position 7 with Ile, Abu, or Val;(iv) substitution of Gln at position 20 with Ser, Thr, Ala, Aib, Arg, orLys; (v) substitution of Met at position 27 with Leu or Nle; (vi)deletion of amino acids at positions 28-29; (vii) deletion of the aminoacid at positions 29; (viii) addition of the amino acid sequenceGPSSGAPPPS (SEQ ID NO: 1610) to the C-terminus; (ix) addition of theamino acid sequence GPSSGAPPPSX (SEQ ID NO: 1450) to the C-terminus,wherein X is any amino acid; and (x) a combination thereof; and, whereinQ exhibits glucagon agonist activity; or, (III) a glucagon relatedpeptide of SEQ ID NO: 1601, with the following modifications: (a) anamino acid modification at position 1 that confers GIP agonist activity;and, (b) a modification selected from the group consisting of: (i) alactam bridge between the side chains of amino acids at positions i andi+4 or between the side chains of amino acids at positions j and j+3,wherein i is 12, 13, 16, 17, 20 or 24, and wherein j is 17, and (ii)one, two, three, or all of the amino acids at positions 16, 20, 21, and24 is substituted with an α,α-disubstituted amino acid; and, (c) 1-10further amino acid modifications; and, wherein Q exhibits activity atthe GIP receptor; or, (IV) the amino acid sequence:X₁-X₂-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X₃-Ser-X4-Tyr-Leu-X₅-X₆-X₇-X₈-Ala-X₉-X₁₀-Phe-X₁₁-X₁₂-Trp-Leu-X₁₃-X₁₄-X₁₅(SEQ ID NO: 55), or an analog thereof, wherein said analog differs fromSEQ ID NO: 55 by 1 to 3 amino acid modifications, selected frompositions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, and 25,wherein: X₁ is His, D-His, (Des-amino)His, hydroxyl-His, acetyl-His,homo-His, or alpha, alpha-dimethyl imidazole acetic acid (DMIA),N-methyl His, alpha-methyl His, or imidazole acetic acid; X₂ is Ser,D-Ser, Ala, D-Ala, Val, Gly, N-methyl Ser, aminoisobutyric acid (Aib) orN-methyl Ala; X₃, X₄, X₅, X₁₀, X₁₁, and X₁₄ are, individually, any aminoacid; X₆ is Ser, Glu, Gln, homoglutamic acid or homocysteic acid; X₇ isArg, Gln, Lys, Cys, Orn, homocysteine or acetyl phenylalanine; X₈ isArg, Ala, Lys, Cys, Orn, homocysteine or acetyl phenyalanine; X₉ is Gln,Lys, Arg, Orn or Citrulline; X₁₂ is Ala, Gln, Glu, Lys, Cys, Orn,homocysteine or acetyl phenyalanine; X₁₃ is Met, Leu or Nle; X₁₅ is Thr,Gly, Lys, Cys, Orn, homocycsteine or acetyl phenyalanine; and wherein Qoptionally comprises one of the following modifications: (a) deletion ofamino acids at positions 28-29; (b) deletion of the amino acid atpositions 29; (c) addition of the amino acid sequence GPSSGAPPPS (SEQ IDNO: 1610) to the C-terminus; and (d) addition of the amino acid sequenceGPSSGAPPPSX (SEQ ID NO: 1450) to the C-terminus, wherein X is any aminoacid; and (e) a combination thereof; or, (V) an amino acid sequence thatdiffers from SEQ ID NO: 1601 by no more than ten amino acidmodifications, comprising: (a) one or more amino acid substitutions withAib at positions 16, 20, 21, and/or 24, and (b) an amino acidmodification at position 1 and/or 2 that provides reduced susceptibilityto cleavage by dipeptidyl peptidase IV, and wherein Q optionallycomprises one or more of the following modifications: (i) deletion ofamino acids at positions 28 and 29, (ii) deletion of the amino acid atposition 29, (iii) addition of the amino acid sequence GPSSGAPPPS (SEQID NO: 1610) to the C-terminus, (iv) addition of the amino acid sequenceGPSSGAPPPSX (SEQ ID NO: 1450) to the C-terminus, wherein X is any aminoacid, (v) substitution of the C-terminal carboxyl group with an amide orcarboxylic ester, and wherein Q exhibits GLP-1 agonist activity andglucagon agonist activity.
 7. The compound of claim 6, wherein in (I)the 1-10 further amino acid modifications are selected from the groupconsisting of: (i) substitution of serine at position 2 with D-Ser, Ala,D-Ala, Gly, N-methyl-Ser, Aib, Val, or ε-amino-N-butyric acid; (ii)substitution of serine at position 16 with Glu, Gln, homoglutamic acid,homocysteic acid, Thr, Gly, or Aib; (iii) substitution of glutamine atposition 20 with Ser, Thr, Ala, Lys, Citrulline, Arg, Orn, or Aib; (iv)substitution of methionine at position 27 with Leu; (v) substitution ofarginine at position 28 with Ala; (vi) substitution of threonine atposition 29 with Gly; (vii) a conservative substitution at any ofpositions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 24, 27,28, and 29; (viii) addition of 1-21 amino acids to the C-terminus (ix)addition of the amino acid sequence GPSSGAPPPS (SEQ ID NO: 1610) to theC-terminus, (x) addition of the amino acid sequence GPSSGAPPPSX (SEQ IDNO: 1450) to the C-terminus, wherein X is any amino acid, (xi)substitution of the C-terminal carboxyl group with an amide orcarboxylic ester, and (xii) a combination thereof.
 8. The compound ofclaim 1, wherein L-Y is covalently conjugated to the N-terminus,C-terminus, or an amino acid side chain of Q that corresponds toposition 10, 30, 37, 38, 39, 40, 41, 42, or 43 of native glucagon (SEQID NO: 1601) or a peptide comprising native glucagon.
 9. A prodrug ofthe compound claim 1 comprising the structure A-B, wherein A is an aminoacid or a hydroxy acid; B is an N-alkylated amino acid linked to Qthrough an amide bond between a carboxyl moiety of B and an amine of Q;A, B, or the amino acid of Q to which A-B is linked is a non-coded aminoacid, further wherein the chemical cleavage half-life (t_(1/2)) of A-Bfrom Q is at least about 1 hour to about 1 week in PBS underphysiological conditions.
 10. The compound of claim 9, wherein A-Bcomprises the structure:

wherein (a) R¹, R², R⁴ and R⁸ are independently selected from the groupconsisting of H, C1-C18 alkyl, C2-C18 alkenyl, (C1-C18 alkyl)OH, (C1-C18alkyl)SH, (C2-C3 alkyl)SCH₃, (C1-C4 alkyl)CONH₂, (C1-C4 alkyl)COOH,(C1-C4 alkyl)NH₂, (C1-C4 alkyl)NHC(NH₂ ⁺)NH₂, (C0-C4 alkyl)(C3-C6cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocyclic), (C0-C4 alkyl)(C6-C10aryl)R⁷, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C12 alkyl(W1)C1-C12alkyl, wherein W1 is a heteroatom selected from the group consisting ofN, S and O, or (i) R¹ and R² together with the atoms to which they areattached form a C3-C12 cycloalkyl or aryl; or (ii) R⁴ and R⁸ togetherwith the atoms to which they are attached form a C3-C6 cycloalkyl; (b)R³ is selected from the group consisting of C1-C18 alkyl, (C1-C18alkyl)OH, (C1-C18 alkyl)NH₂, (C1-C18 alkyl)SH, (C0-C4alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocyclic), (C0-C4alkyl)(C6-C10 aryl)R⁷, and (C1-C4 alkyl)(C3-C9 heteroaryl) or R⁴ and R³together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring; (c) R⁵ is NHR⁶ or OH; (d) R⁶ is H, C1-C8 alkylor R⁶ and R² together with the atoms to which they are attached form a4, 5 or 6 member heterocyclic ring; and (e) R⁷ is selected from thegroup consisting of H and OH.
 11. The compound of claim 1, furthercomprising an amino acid side chain on Q covalently attached to an acylgroup or an alkyl group via an alkyl amine, amide, ether, ester,thioether, or thioester linkage, which acyl group or alkyl group isnon-native to a naturally occurring amino acid.
 12. The compound ofclaim 11, wherein the amino acid to which the acyl or alkyl group isattached is at a position corresponding to position 10, 20, 24, 30, 37,38, 39, 40, 41, 32, or 43, or the C-terminal amino acid.
 13. Thecompound of claim 12, wherein the acyl group or the alkyl group isattached to the side chain of the amino acid through a spacer.
 14. Thecompound of claim 1, wherein Q is covalently attached to one or moreheterologous moieties.
 15. The compound of claim 1 wherein Q is selectedfrom the group consisting of SEQ ID NOs: 1-564, 566-570, 573-575, 577,579-580, 585-612, 616, 618-632, 634-642, 647, 657-692, 694-695, 715-718,722, 724-725, 729, 731-760, 801-878, 883-919, 1001-1275, 1301-1371,1401-1518, and 1601-1650.
 16. A pharmaceutical composition comprisingthe compound of claim 1 and a pharmaceutically acceptable carrier.
 17. Amethod for treating a disease or medical condition in a patient, whereinthe disease or medical condition is selected from the group consistingof metabolic syndrome, diabetes, obesity, liver steatosis, and aneurodegenerative disease, comprising administering to the patient thepharmaceutical composition of claim 16 in an amount effective to treatthe disease or medical condition.
 18. The compound of claim 1 wherein Yis a β3 adrenergic receptor agonist.
 19. The compound of claim 18wherein the β3 adrenergic receptor agonist is solabegron(3′-[(2-{[R2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}ethyl)amino]biphenyl-3-carboxylicacid).
 20. The compound of claim 18 wherein Q is exendin-4 (SEQ ID NO:1618).
 21. The compound of claim 1 wherein the G protein-coupledreceptor ligand activates the G protein-coupled receptor with an EC₅₀ ofabout 100 nM or less.